KIAA1199, a deafness gene of unknown function,is a new hyaluronan binding protein involvedin hyaluronan depolymerizationHiroyuki Yoshidaa,1, Aya Nagaokaa, Ayumi Kusaka-Kikushimaa, Megumi Tobiishia, Keigo Kawabataa, Tetsuya Sayoa,Shingo Sakaia, Yoshinori Sugiyamaa, Hiroyuki Enomotob, Yasunori Okadac,1, and Shintaro Inouea,1

aInnovative Beauty Science Laboratory, Kanebo Cosmetics, Inc., Kotobuki-cho, Odawara-shi, Kanagawa 250-0002, Japan; bDepartment of OrthopaedicSurgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-0016, Japan; and cDepartment of Pathology, School of Medicine, Keio University,Shinjuku-ku, Tokyo 160-0016, Japan

Edited by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved February 19, 2013 (received for review October 5, 2012)

Hyaluronan (HA) has an extraordinarily high turnover in physiolog-ical tissues, and HA degradation is accelerated in inflammatory andneoplastic diseases. CD44 (a cell surface receptor) and two hyalur-onidases (HYAL1 and HYAL2) are thought to be responsible for HAbinding and degradation; however, the role of these molecules inHA catabolism remains controversial. Herewe show that KIAA1199,a deafness gene of unknown function, plays a central role in HAbinding and depolymerization that is independent of CD44 andHYAL enzymes. The specific binding of KIAA1199 to HA was dem-onstrated in glycosaminoglycan-binding assays. We found thatknockdown of KIAA1199 abolished HA degradation by human skinfibroblasts and that transfection of KIAA1199 cDNA into cells con-ferred the ability to catabolize HA in an endo-β-N-acetylglucosami-nidase–dependent manner via the clathrin-coated pit pathway.Enhanced degradation of HA in synovial fibroblasts from patientswith osteoarthritis or rheumatoid arthritis was correlated with in-creased levels of KIAA1199 expression and was abrogated byknockdownof KIAA1199. The level of KIAA1199 expression in unin-flamed synovium was less than in osteoarthritic or rheumatoidsynovium. These data suggest that KIAA1199 is a unique hyalad-herin with a key role in HA catabolism in the dermis of the skin andarthritic synovium.

extracellular matrix | hyaluronic acid | hyaluronate | hearing loss

Hyaluronan (HA) is a high molecular weight, linear glycos-aminoglycan (GAG) composed of only two sugars: β-(1,3)-

linked-D-glucuronic acid and β-(1,4)-linked-N-acetyl-D-glucos-amine. HA is ubiquitously present as a major constituent of theextracellular matrix (ECM) in vertebrate tissues, providing struc-tural and functional integrity to cells and organs. Although manyorgans maintain high concentrations of HA, skin contains ap-proximately half the total body HA (1). HA is rapidly depoly-merized within tissues, from extralarge native molecules of 1,000–10,000 kDa, to intermediate-size fragments of 10–100 kDa presentin the extracellular milieu (2). Approximately one-third of totalbody HA is replaced daily, and the skin is a major determinantorgan for HA turnover, with a metabolic half-life of 1–1.5 d (2).HA degradation is enhanced under certain pathological conditionsand its lower molecular weight products are commonly detected indiseases, such as arthritis and cancers (3–5). The reduced averagemolecular weight of HA (as low as 200 kDa) in synovial fluids frompatients with osteoarthritis (OA) or rheumatoid arthritis (RA)leads to decreased synovial viscosity and is associated with synovialinflammation (6). In addition, much lower molecular weight HAfragments (∼20 kDa) are known to stimulate neovascularizationand facilitate tumor cell motility and invasion (5, 7, 8).There are six human hyaluronidase-related genes clustered on

two chromosomal loci, 3p21.3 (HYAL1, HYAL2, and HYAL3) and7q31.3 (HYAL4, HYALP1, and SPAM1) (9). However, becauseHYALP1 is a pseudogene (9), and HYAL4 and SPAM1 have re-stricted expression patterns, HYALP1, HYAL4, and SPAM1 are

unlikely to have major roles in constitutive HA degradation in vivo.HYAL3 has a restricted expression pattern (9) and its ability todegrade HA is questionable (10). Therefore, HYAL1 and HYAL2are most likely to have key roles in degrading HA.One current model suggests that high molecular weight HA is

tethered to cell surfaces by CD44 (a HA receptor) concentrated incaveolin-rich lipid rafts, and then cleaved by HYAL2 into in-termediate-size fragments in acidic microenvironments created bythe Na+-H+ exchanger (11). The intermediate-size fragments aredegraded to oligosaccharides within cells by lysosomal HYAL1 incoordination with lysosomal β-glucuronidase and β-N-acetyl-glu-curosaminidase (12). However, this model is insufficient to explainthe rapid catabolism of HA in vivo. First, neither HYAL1 norHYAL2 are expressed in the brain (9, 13), a major organ con-taining large amounts of HA. This finding suggests that othermolecules degrade HA in the brain. Second, HYAL2 has little orno hyaluronidase activity (14), and the finding that HYAL1degrades HA into oligosaccharides of∼0.8 kDa intracellularly (10,12) is inconsistent with the presence of HA catabolites of 10–100kDa in tissues (2). Third, mice deficient in the HYAL1 or HYAL2gene do not show significant accumulation of HA within tissues(15, 16). Finally, the evidence for HA degradation by HYAL1 andHYAL2 was obtained in a breast carcinoma MDA-MB231 cellline (11) and in cells stably transfected with these genes (10); thus,only limited evidence for the direct involvement of HYAL1 andHYAL2 in HA degradation in cells such as fibroblasts is available.Collectively, these lines of evidence reveal that HA degradation bythe HYAL enzymes remains elusive, and that new HA-degrada-tion pathways independent of the HYAL1, HYAL2, and CD44system may exist.In the present study, we first tested the involvement of HYAL1,

HYAL2, and CD44 in HA depolymerization in normal humanskin fibroblasts, and found that knockdown of these genes withsiRNAs did not abrogate HA depolymerization. This resultprompted us to investigate new mechanisms for HA degradation.Using microarray analysis, we screened genes whose expressionlevels paralleled the extent of HA depolymerization in culturedskin fibroblasts under stimulated conditions. Intriguingly, our dataprovided unique evidence that a deafness gene of unknown func-tion, known as KIAA1199 (17), has a key role in the binding anddepolymerization of HA, and that this activity is independent of

Author contributions: H.Y., H.E., Y.O., and S.I. designed research; H.Y., A.N., A.K.-K., M.T.,K.K., and S.S. performed research; H.E. and Y.O. contributed new reagents/analytic tools;H.Y., A.N., K.K., T.S., S.S., Y.S., H.E., Y.O., and S.I. analyzed data; and H.Y., Y.O., and S.I.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: yoshida.hiroyuki@kanebocos.co.jp,okada@z6.keio.jp, or inoue.shintarou@kanebocos.co.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1215432110/-/DCSupplemental.

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CD44 and HYAL enzymes. We also demonstrate that KIAA1199is expressed predominantly by dermal fibroblasts in normal skinand is overexpressed by synovial fibroblasts and tissues fromarthritic joints.

ResultsCD44 and HYAL Enzymes Are Dispensable for HA Degradation in CulturedSkin Fibroblasts. Human embryonic skin fibroblasts (Detroit 551cells) degraded exogenous high molecular weight [3H]-labeledHA of >1,000 kDa ([3H]HA) to intermediate size fragments withmolecular weights ranging from 10 kDa to 100 kDa, and accu-mulated the catabolites extracellularly (Fig. 1A). The fibroblastsselectively digested fluoresceinamine-labeled HA of 1,760 kDa(FA-HA H1), but not other FA-labeled GAGs, such as chon-droitin sulfate A, C, and D (FA-CSA, FA-CDC, and FA-CSD),dermatan sulfate (FA-DS), heparin (FA-Hep), or heparan sulfate(FA-HS) (Fig. S1), suggesting that the depolymerizing machineryof the fibroblasts is specific for HA. To study the involvement ofCD44 and the enzymes HYAL1 and HYAL2 in HA depolymeri-zation, we examined expression of these molecules and found thatskin fibroblasts express CD44 and HYAL2 but not HYAL1 (Fig.1B). Interestingly, knockdown of CD44 and HYAL2 using twodifferent siRNAs specific to these molecules showed no effect onHA depolymerization (Fig. 1 C and D). These data suggest thatthe HA-degrading machinery present in Detroit 551 cells is in-dependent of CD44 and HYAL2 or HYAL1.

KIAA1199 Is Required for HA Depolymerization and Expressed byDermal Fibroblasts in Normal Skin. To identify molecules involvedin the HA depolymerization, we monitored changes in depolymeri-zation in Detroit 551 skin fibroblasts following stimulation withIL-1α, IL-1β, TGF-α, TGF-β1, epidermal growth factor, hepato-cyte growth factor, IFN-γ, TNF-α, or histamine, and found thatHA depolymerization is strikingly up-regulated and down-regu-lated only by treatment with histamine and TGF-β1, respectively(Fig. 1E). Thus, we searched for genes associated with HA de-polymerization using microarray analysis and identified 25 genesthat were up-regulated by histamine and down-regulated by TGF-β1 (Table S1). We then used siRNA to knock down the 25 genesand discovered that transfection of two different siRNAs targetingthe KIAA1199 gene abolished HA depolymerization in Detroit551 cells (Fig. 1F and Fig. S2A). Similar results were obtained withother human skin fibroblast cell lines, including HS27 (neonatalskin fibroblasts) and NHDF-Ad (adult skin fibroblasts) (Fig. S2 Band C). The HA depolymerizing activity in Detroit 551 cellsshowed an apparent Vmax of 370 μg/105 cells/72 h and Km of 1,480μg/mL. Histamine treatment showed a 3.8-fold increase in Vmax(1,370 μg/105 cells/72 h) without affecting Km (1,500 μg/mL) (Fig.1G). Under stimulation with histamine, the mRNA and proteinexpression of KIAA1199 was significantly increased by 3.7-fold(0.37 ± 0.02 vs.1.37 ± 0.04, control vs. histamine; n = 3; P < 0.001)(Fig. 1H) and 4.2-fold (0.31± 0.10 vs. 1.31± 0.10; n= 3; P< 0.001)respectively (Fig. 1I), whereas TGF-β1 reduced the expression toa negligible level (Fig. 1 H and I). These results show thatKIAA1199 expression is essential for HA depolymerization in skinfibroblasts and the amount of KIAA1199 protein directly deter-mines the velocity of HA depolymerization. In situ hybridization(Fig. 2A) and immunohistochemistry (Fig. 2B) showed thatKIAA1199 is expressed predominantly by dermal fibroblasts innormal human skin.

Cells Transfected with KIAA1199 cDNA Acquire HA-Degrading Capability.To further examine the activity of KIAA1199 in HA depolymeri-zation, we transfected HEK293 (human embryonic kidney) and

Fig. 1. HA degradation via KIAA1199 and regulation of KIAA1199 expres-sion by histamine and TGF-β1 in human skin fibroblasts. (A) Detroit 551 skinfibroblasts were cultured with [3H]HA for 48 h and HA fragments in theculture medium were examined by size-exclusion chromatography. (B) Ex-pression of CD44, HYAL2, and HYAL1 by RT-PCR. GAPDH, a loading control. (Cand D) CD44 and HYAL2 were knocked down by treating cells with siRNAs toCD44 or HYAL2. For controls, the cells were transfected with control non-silencing siRNA (Control siRNA). Cells with siRNA were cultured with [3H]HAfor 48 h, and degraded HA was examined by size-exclusion chromatography.Knockdown efficiency for CD44 and HYAL2 was evaluated by immunoblot-ting (Insets). GAPDH, a loading control. Representative data for two siRNAsare shown. (E) Effect of histamine and TGF-β1 on HA depolymerization. Cellswere treated with or without histamine or TGF-β1 and cultured with [3H]HAfor 48 h. The HA-degrading activity was analyzed by chromatography. Con-trol, untreated cells. (F) Abrogation of HA-degrading activity by knockdownof KIAA1199 with two different siRNAs specific for KIAA1199. The cells werecultured with [3H]HA for 48 h, and HA degradation was determined. (Inset)Immunoblotting for KIAA1199 and GAPDH (a loading control). (G) Kineticstudy of HA degradation by cells treated with or without histamine for 72 h.Control and Histamine, cells treated with vehicle alone and histamine, re-spectively. (H and I) The expression levels of KIAA1199 mRNA and protein incells treated with histamine, TGF-β1 or vehicle alone (Cont) for 24 h. Levels ofmRNA and protein expression were measured by real-time PCR and immu-noblotting. Values (relative mRNA expression, fold KIAA1199 to GAPDH)represent mean ± SD (n = 3). The Dunnett test was used for statistical analysis.The protein expression levels (ratio of KIAA1199 to GAPDH) were estimated

by using densitometric scanning Multi Gauge v.2.1 (Fuji Film). A represen-tative finding of three different experiments is shown.

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COS-7 (monkey kidney fibroblast) cell lines—neither of whichhave HA-degrading activity—with KIAA1199 cDNA. KIAA1199transfectants reduced [3H]HA to intermediate size fragments of10–100 kDa, releasing the catabolites into the medium as seenwith skin fibroblasts, whereas mock transfectants showed negligi-ble activity (Fig. 3A and B).We then prepared stable transfectantsof KIAA1199 in HEK293 cells (KIAA1199/HEK293 cells) andshowed that KIAA1199/HEK293 cells selectively digest FA-HAH1, but not other FA-GAGs (FA-CSA, FA-CDC, FA-CSD, FA-DS, FA-Hep, and FA-HS) (Fig. S3). In addition, KIAA1199/HEK293 cells degraded FA-HA species with different averagemolecular weights (FA-M1, 907 kDa; FA-L1, 197 kDa; FA-S1, 56kDa; FA-T1, 28 kDa; FA-U1, 9.8 kDa) (Fig. S3). Determinationof the terminal sugars on the fragments showed that the reducingand nonreducing terminal sugars were N-acetylglucosamine andglucuronic acid, respectively (Fig. 3 C and D), indicating thatcleavage occurs at the β-endo-N-acetylglucosamine bonds. Im-portantly, we found that additional, transient expression ofHYAL1 or HYAL2 in KIAA1199/HEK293 cells did not enhanceHA degradation (Fig. S4).

HA Is Catabolized by KIAA1199 via the Clathrin-Coated Pit Pathway.Accumulated lines of evidence have shown that HA degradationoccurs extracellularly and via receptor-mediated endocytosis (2,10–12, 18–20). HARE (HA receptor for endocytosis; also calledStabilin-2) and CD44 are thought to be associated with clathrin-coated pits and caveolae, respectively (2, 11). Because KIAA1199appeared to be located in the cytoplasm and on the cell mem-brane, as shown in Fig. 2B, we assessed the possible involvement ofclathrin-coated pits and caveolae for their roles in HA degrada-tion. When expression of the clathrin heavy chain (CHC) andα-adaptin subunit of AP-2, an adaptor protein complex function-ing as a major organizer of clathrin coats, was knocked down bysiRNAs in KIAA1199/HEK293 cells, HA degradation was re-duced (Fig. 4 A and B). In contrast, knockdown of caveolin-1caused no changes in HA degradation (Fig. 4C). Immunoprecip-itation with anti-KIAA1199 antibody showed that CHC copreci-pitates with KIAA1199 (Fig. 4D). Because these results suggestthat the clathrin-coated pit pathway is most likely involved inKIAA1199-mediated HA depolymerization, we further studiedthe effects of inhibitors on HA degradation. These effects in-cluded inhibitors for receptor recycling (monensin), endosome-lysosome system acidification (NH4Cl), a vacuolar (H+)-ATPase(bafilomycin A1), dynamin, a GTPase implicated in endocytosisand scission of newly formed vesicles (dynasore), and polymeri-zation of microtubules and trafficking from early to late endosome(nocodazole) (19). As shown in Fig. 4E, treatment of KIAA1199/

HEK293 cells with these chemicals, except for nocodazole,inhibited HA depolymerization, suggesting that HA is depoly-merized in acidic compartments before endosome-lysosome fu-sion (e.g., clathrin-coated vesicles or early endosomes). The ideathat HA depolymerization is unlikely to occur in lysosomes issupported by the observations that additional expression of lyso-somal HYAL1 or HYAL2 in KIAA1199/HEK293 cells did notaffect HA depolymerization (Fig. S4). By immunohistochemistry,KIAA1199 was localized mainly to the vesicles in the periphery ofDetroit 551 skin fibroblasts (Fig. S5 A and B). Double immuno-staining of KIAA1199 and CHC in KIAA1199/HEK293 cellsshowed that signals of both molecules are detected in a vesicularpattern, and KIAA1199 is localized closely to CHC in somevesicles (Fig. S5 C and D). When localization of high molecularweight HA added to skin fibroblasts and KIAA1199/HEK293 cellswas examined by confocal microscopy, HA was observed in theperiphery of these cells, showing a vesicular pattern, but no fluo-rescence was shown by incubation with Streptomyces hyaluroni-dase-digested HA (Fig. S5E–G). Importantly, [3H]HA was almostcompletely recovered in the medium after addition to KIAA1199/HEK293 cells, and intracellular HA accumulation was not ob-served (Table S2). In addition, [3H]HA was not depolymerizedwhen it was incubated with conditioned media from KIAA1199/HEK293 cells. All these results suggest that KIAA1199-mediatedHA depolymerization may occur through rapid vesicle endocytosisand recycling without intracytoplasmic accumulation or digestionin lysosomes.

KIAA1199 Has HA-Specific Binding Capability. The interaction be-tween KIAA1199 protein and GAGs was examined by immuno-blotting for KIAA1199 in GAG precipitates from the KIAA1199/HEK293 cell lysates after incubation with GAGs. As shown in Fig.4F, KIAA1199 was selectively coprecipitated with HA (Fig. 4F,Upper), and also coprecipitated with HA species of varying aver-age molecular weights including HA-H2 (1,452 kDa), HA-M2

Fig. 2. Expression of KIAA1199 by dermal fibroblasts in normal human skin.(A) The mRNA expression of KIAA1199was examined by in situ hybridizationusing antisense (Left) and sense (Right) RNA probes. (B) KIAA1199 proteinexpression was analyzed by immunohistochemistry with anti-KIAA1199 Ab(Left) and control rat IgG2aκ (Right). (Insets) High-power views of the boxedareas. Arrows, KIAA1199+ cells. (Scale bars, 50 μm; Insets, 20 μm.) Repre-sentative data from three subjects are shown.

Fig. 3. HA depolymerization by KIAA1199 transfectants and determinationof HA cleavage sites. (A and B) HEK293 and COS-7 cells were transientlytransfected with empty vector (Mock) or vector containing KIAA1199 cDNA,and then incubated with [3H]HA for 24 h. HA depolymerization was exam-ined by size-exclusion chromatography. Expression of KIAA1199 protein inMock and KIAA1199 transfectants was assessed by immunoblotting (Insets).(C and D) Determination of the reducing and nonreducing terminal sugars ofdepolymerized HA. HPLC pattern of pyridylaminated N-acetylglucosamine(PA-GLcNA) obtained from HA depolymerized by KIAA1199/HEK293 cells (C).Sephadex G-25 column chromatogram of depolymerized [3H]HA after in-cubation with β-N-acetylglucosaminidase (○) or β-glucuronidase followed byincubation with β-N-acetylglucosaminidase (●) (D).

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(1,039 kDa), HA-L2 (219 kDa), HA-S2 (52 kDa), and HA-T2 (28kDa) (Fig. 4F, Lower). In addition, KIAA1199 in KIAA1199/HEK293 cell lysates could bind to HA-Sepharose beads (Fig. 4G,

Upper), and this binding was competitively blocked by pre-incubation of KIAA1199/HEK293 cell lysates with soluble HA-H2,but not other GAGs (Fig. 4G, Lower). Note, however that HA-degrading activity was not detected in cell lysates from KIAA1199/HEK293 cells. Recombinant KIAA1199 protein expressed ina wheat-germ cell-free expression system showed no definitive HAdegrading activity at a pH range between 4.0 and 7.0 (Fig. S6).

KIAA1199 Is Overexpressed by Synovial Fibroblasts and Synovial TissuesfromOA and RAPatients.To assess the possible role of KIAA1199 inhuman disease, we next examined HA-degrading activity in cul-tured synovial fibroblasts. The activity appeared higher in OA andRA synovial fibroblasts compared with normal synovial fibroblasts(Fig. 5A and Fig. S7) and closely correlated with the expressionlevels of KIAA1199 mRNA and protein (Fig. 5B). Importantly,knockdown of KIAA1199 by siRNA reduced the HA-degradingactivity to almost negligible levels (Fig. 5A and Fig. S7). These datasuggest that KIAA1199 is essential for HA degradation in synovialfibroblasts and that overexpression of KIAA1199 elicits enhancedHA degradation in OA and RA synovial cells. Real-time PCRanalysis showed that KIAA1199 gene expression tended to behigher in OA synovial tissues (P = 0.088) and was significantlyhigher in RA synovial tissues (P < 0.05) compared with non-inflammatory synovial tissue (Fig. 5C). In situ hybridization andimmunohistochemistry showed that KIAA1199 is expressedmainly by synovial lining and some sublining cells of RA synovialtissues, whereas only background signal was observed with a con-trol sense probe and nonimmune IgG (Fig. 5 D and E).

Missense Mutations of KIAA1199 in Hearing-Loss Patients Reduce HADegradation. Because four missense mutations in KIAA1199 havebeen associated with hearing loss (17), the effect of KIAA1199mutations on HA-degrading activity was examined. As shown inFig. S7, four mutant KIAA1199 proteins (R187C, R187H,H783R,and V1109I) and wild-type protein were expressed at similar levelsby transient transfection in HEK293 cells. Cells expressing R187Cand R187H mutants showed marked reductions in HA degradingactivity compared with the H783R and V11091 mutants and wild-type proteins.

DiscussionTwo hyaluronidases (HYAL1 and HYAL2) and CD44 are thoughtto have key roles in HA degradation. However, our present experi-ments on expression of these molecules and KIAA1199, modu-lation of their expression in normal skin and arthritic synovialfibroblasts, and transfection of KIAA1199 cDNA failed to confirmthat notion, and instead provide unique data showing that HAdepolymerization mediated by KIAA1199 occurs independentlyof CD44/HYALs. We found that: (i) normal human skin and ar-thritic synovial fibroblasts degrade HA into intermediate-sizedfragments, dependent on the expression of KIAA1199; (ii)transfection of KIAA1199 cDNA confers the ability to specificallybind to and degrade HA; (iii) overexpression of HYAL1 orHYAL2 in KIAA1199 transfectants does not increase HA de-polymerization; and (iv) HA depolymerization by KIAA1199involves the clathrin-coated pit pathway, rather than the caveolaepathway via CD44 and HYAL2.Extralarge (1,000–10,000 kDa) native HA molecules within

tissues are initially depolymerized into intermediate fragments of10–100 kDa (2). Most fragments are then released from the ECM,drained into lymphatic vessels, and catabolized within the lymphnodes (2). The remaining HA fragments reach the circulation andare fully degraded by an acid-active hyaluronidase in lysosomesfollowing HARE-mediated clathrin-coated pit endocytosis, pre-dominantly in the liver, kidney, and spleen (2), where bothHYAL1 and HYAL2 are highly expressed (9, 13). Patients withmucopolysaccharidosis (MPS) IX caused by HYAL1 deficiencyin humans, and HYAL2 knockout mice, show elevated levels of

Fig. 4. Clathrin-specific HA depolymerization and HA-specific binding ofKIAA1199 in KIAA1199/HEK293 cells. (A–C) CHC (A), α-adaptin (B), and caveolin-1(C) were knocked down by siRNAs to each gene in KIAA1199/HEK293 cells. Forcontrols, cells were transfected with control nonsilencing siRNA. The cells wereincubatedwith [3H]HA for 6 h, andHA fragments in themediawere analyzedbysize-exclusion chromatography. Efficiency of the knockdown was evaluated byimmunoblotting (Insets). Representative data from two siRNAs are shown. Notethat knockdown of CHC and α-adaptin but not caveolin-1 decreased HA de-polymerization. (D) Coimmunoprecipitation of CHC with KIAA1199. Cell lysateswere immunoprecipitated with control IgG2aκ or anti-KIAA1199 antibody, fol-lowed by immunoblotting (IMB) for KIAA1199 and CHC. Input is shown in theUpper panel. IP, immunoprecipitation. (E) Effects of inhibitors on HA depo-lymerization. KIAA1199/HEK293 cells were incubated for 3 h in the absence orpresence of monensin, NH4Cl, bafilomycin A1, dynasore, or nocodazol, followedby additional incubation for 6 h with [3H]HA. HA depolymerization was de-termined by chromatography. (F) GAG-binding assay for KIAA1199 protein. Celllysates of KIAA1199/HEK293 cells were incubated with H2O (negative control) orunlabeled HA (HA-H2), chondroitin sulfate A, C, and D (CSA, CSC, and CSD), der-matan sulfate (DS), heparin (Hep), and heparan sulfate (HS) (Upper), or HA-H2,HA-M2, HA-L2, HA-S2 or HA-T2 (Lower). The samples were precipitated withcetylpyridium chloride and analyzed by NuPAGE and immunoblotting with anti-KIAA1199 antibody. (G) KIAA1199 binding to HA-Sepharose. Cell lysates wereincubated with control or HA-coupled Sepharose 4B beads (Upper), and werepreincubated with H2O (control) or GAGs before application to HA-Sepharose(Lower). Bound materials to the beads were eluted with NuPAGE LDS sample-loading buffer, and analyzed by immunoblotting with anti-KIAA1199 antibody.

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HA in the plasma (13, 16). Lysosomal accumulation of HA is alsoknown in MPS IX patients within macrophages and fibroblasts (13)and HYAL1 knockout mice within chondrocytes (15). These datasuggest that HYAL1 and HYAL2 contribute to HA degradationvia HARE- and CD44-mediated endocytosis in these organs. How-ever, these HYAL enzymes are not expressed in the brain (9, 13),indicating a requirement for an alternative pathway in the brain.KIAA1199 is expressed in a wide range of normal human tissues,including brain, lung, pancreas, testis, and ovary, but its expression isnotably absent in the liver, kidney, and spleen (21). Therefore, itseems likely that KIAA1199may have amore ubiquitous role inHAcatabolism in tissues that do not express HYAL1 or HYAL2, such

as the brain. Moreover, our data show that KIAA1199, but not theHYAL enzymes, is involved in HA degradation in normal skinfibroblasts and arthritic synovial fibroblasts. The differential roles ofKIAA1199 and the CD44/HYALs system in degrading HA in othertissues remain to be clarified in future work.The KIAA1199 protein sequence shows no substantial homol-

ogy to other molecules, including HYAL enzymes, HA-bindingproteins, and bacterial hyaluronidases (9, 12), and lacks HA-linkmodules (22), B(X7)B HA-binding motifs (23), and the transmem-brane domain (21). However, the protein has two GG domains,consisting of two well-conserved Gly residues, oneG8 domain thatcontains eight conserved Gly residues in five β-strand pairs (24,25), four PbH1 domains (26), and seven predictedN-glycosylationsites (26). In the present study, we have shown that mutations ofthe ARG187 residue (R187C and R187H) located in the GG do-main eliminate HA-degrading activity. No information is availablefor the function of the GG domain at present, but the G8 domainhas predicted roles in extracellular ligand binding (25). In addi-tion, the cellular role of PbH1 domain is supposed to be poly-saccharide hydrolysis according to the InterPro member databaseSMART (www.ebi.ac.uk/interpro). Thus, it is possible to speculatethat the GG and G8 domains and PbH1 domains are responsiblefor KIAA1199-mediated binding and depolymerization of HA.LikeHYAL2 (14), cell lysates of KIAA1199 stable transfectants orrecombinant KIAA1199 protein lacked HA depolymerizing ac-tivity. This lack could reflect the need for a specific conformationalchange in KIAA1199 that is conferred by its microenvironment orinteractions with other molecules. Because our study showed thatKIAA1199 is located near the CHC+ small vesicles, andKIAA1199-mediated HA degradation is dependent on vacuolar (H+)-ATPaseand dynamin, the acidic, dynamic and energy-coupling microen-vironment may be critical for KIAA1199 activity. Alternatively, wecannot exclude the possibility that KIAA1199 may function as anadaptor molecule for an unknown hyaluronidase. It is well knownthat free radicals randomly attack HA chains, generating HA frag-ments of various sizes (27). However, our finding that KIAA1199can mediate HA cleavage at β-endo-N-acetylglucosamine bondsshows that HA degradation by KIAA1199 is independent of free-radical activity.The study presented here shows thatKIAA1199 gene expression

is up-regulated and down-regulated by histamine and TGF-β1,respectively. A reduction in molecular weight and mass ofHA because of increased HA degradation is commonly observed inarthritic synovial fluids (3, 4) and in the dermis of theUV-damagedskin (28). Under these pathological conditions, locally, overpro-duced histamine is released from mast cells into the extracellularenvironment (29, 30). It is therefore plausible that up-regulationof KIAA1199 expression by histamine enhances HA degradationin these conditions. In contrast, TGF-β1 plays an essential role inthe overproduction of ECM molecules, including HA, underpathophysiological conditions such as wound healing (31). Al-though accumulation of HA within wounded tissues is explainedmainly by enhanced HA synthesis following TGF-β–induced ex-pression of HA synthases 1 and 2 (32), our data suggest that TGF-β1 might simultaneously down-regulate HA degrading activity byreducing KIAA1199 gene expression, which contributes further tothe accumulation of HA.We have shown that the R187C and R187H mutants of

KIAA1199 substantially reduce HA depolymerization. HA ispresent in the inner ear as a molecular filter against intercellularionic leakage (33) and KIAA1199 is highly expressed in the spiralligament of the cochlea (17), which has an important role in thehomeostasis of inner ear fluids (33). Thus, a decline in HA ca-tabolism might affect the static equilibrium of electrolytes andwater in the inner ear fluids, causing hearing impairment. Anotherpossible link between KIAA1199 and genetic disorders is Wernersyndrome. Werner syndrome is an adult-onset progeroid diseasecaused by mutations in the WRN gene encoding a protein with

Fig. 5. KIAA1199-mediated HA degradation in OA and RA synovial fibro-blasts, and expression of KIAA1199 by synovial lining and sublining cells in theRA patients. (A) Synovial fibroblasts from a normal subject (n = 1), OA, and RApatients (n= 3 each) were treatedwith control nonsilencing siRNAor siRNAs forKIAA1199, and incubated with [3H]HA for 48 h. HA depolymerization was an-alyzed by size-exclusion chromatography. Protein expression of KIAA1199 wasassessed by immunoblotting (Insets). Representative data of three OA (OA-2)and RA synovial fibroblasts (RA-3) are shown, and data of other patients arepresented in Fig. S7. Representative data from two siRNAs are shown. (B) Theexpression of KIAA1199 mRNA (Upper) and protein (Lower) in cultured normal(n= 1), OA (n = 3), and RA (n = 3) synovialfibroblasts was analyzed by real-timePCR and immunoblotting. GAPDH, loading control. (C) The expression levels ofKIAA1199 in synovial tissues from the patients with noninflammatory jointdisease (n = 3), OA (n = 10) or RA (n = 8) were determined by real-time PCR.Data are presented as a scatter blot with mean ± SD. Statistical analysis wasdone by the Dunnett test. (D and E) Identification of cells expressing KIAA1199in RA synovial tissue. The expression of KIAA1199 mRNA and protein was de-termined by in situ hybridization using antisense (Left) and sense (Right) RNAprobes (D) and by immunohistochemistry with anti-KIAA1199 Ab (Left) andcontrol rat IgG2aκ (Right) (E). Arrows, KIAA1199-expressing cells. (Scale bars,50 μm.) Representative data of eight subjects are shown.

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helicase and exonuclease activities (34). Interestingly, Wernerpatients show hyperhyaluronic aciduria together with elevated se-rumHA concentration (35) and scleroderma-like skin with an agedappearance, characterized by reduced amounts of HA (36). Skinfibroblasts isolated from a patient with Werner syndrome over-express KIAA1199 (21) and generate small molecular weight HA(37). Although further studies are needed to obtain direct evidence,it is tempting to speculate that enhanced degradation and release ofHA from the skin by fibroblasts overexpressing KIAA1199 maycontribute to the clinical characteristic of Werners syndrome.Previous studies have shown that KIAA1199 is up-regulated ingastric and colorectal cancers and immortalized renal cell carci-nomas, and a splice variant is detected in primary colon adeno-carcinomas, although the functional significance of these findings isnot clear (21, 26, 38). In addition, enhanced expression and activityof HYAL1 andHYAL2/CD44 are reportedly related to growth andmalignancy of tumor cells (11, 39). Because HA fragments of <20kDa can induce inflammation by promoting angiogenesis (7, 8) andcell migration (5), one can postulate that HA fragments generatedby KIAA1199 or HYAL enzymes might contribute to enhancedneovascularization and tumor cell invasion in cancer tissues. How-ever, further detailed studies are necessary to better understand theroles of KIAA1199 and HYAL enzymes in cancer.In summary, the present study provides evidence that KIAA1199

is a unique HA-binding protein, with a role in HA catabolism, thatis independent of CD44 and HYAL enzymes. The expression of

KIAA1199 by dermal fibroblasts in normal skin and by synovialcells in RA synovial tissue suggests that this molecule has key rolesin catabolizing HA in the dermis of healthy skin and the synoviumof arthritis patients. These findings provide fresh insight into ourunderstanding of physiological and pathological HA catabolism,and suggest that therapeutic interventions targeting KIAA1199may be of clinical value.

Materials and Methods[3H]HA of >1,000 kDa was prepared as described in SI Materials and Methods.[3H]HA depolymerization after incubation with cultured cells was determinedby application of the media to a Sepharose CL-2B (GE Healthcare) column.The cDNA of human KIAA1199 was chemically synthesized. A rat monoclonalantibody against human KIAA1199 was raised against the amino acid se-quence CA762RYSPHQDADPLKPRE777, corresponding to amino acids Ala762 toGlu777 of KIAA1199 (GenBank accession no. NM_018689). Immunohisto-chemistry and in situ hybridization studies were done on paraffin sections offormalin-fixed human normal skin and rheumatoid synovium. More detailedinformation is provided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Dr. Y. Endo and Dr. T. Sawasaki of EhimeUniversity for their generous support with a wheat-germ cell-free expressionsystem; Dr. S. Higashiyama of Ehime University and Dr. M. Ito of the New YorkUniversity School of Medicine for discussions; and Dr. A. Fosang of the UniversityofMelbourne,Dr. S.Nakanishi ofOsakaBioscience Institute, andDr.Y.Yamaguchiof Sanford-BurnhamMedical Research Institute for reviewing themanuscript. Thiswork was partially supported by a Grant-in-Aid for Scientific Research (A)24249022 from the Japan Society for the Promotion of Science (to Y.O.).

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6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1215432110 Yoshida et al.

Supporting InformationYoshida et al. 10.1073/pnas.1215432110SI Materials and MethodsCell Cultures.Normal human skin fibroblasts, including Detroit 551(American Type Culture Collection), HS27 (American TypeCulture Collection), and NHDF-Ad (Takara Bio) cells, werecultured in Eagle’s minimum essential medium (MP Biomedicals)supplemented with nonessential amino acids, 1 mM sodium py-ruvate, and 10% (vol/vol) FBS (JRH Biosciences). Human syno-vial fibroblasts from a normal subject (50-y-old male) and patientswith osteoarthritis (OA; 67-y-old female, 61-y-old male, and 77-y-old male) or rheumatoid arthritis (RA; 40-, 55- and 58-y-old fe-males) were purchased from Toyobo. These synovial fibroblasts,HEK293 (DS Pharma Biomedical) and COS-7 (DS Pharma Bio-medical) cell lines were maintained in DMEM (Sigma) supple-mented with 10% (vol/vol) FBS, 100 units/mL penicillin, and 100μg/mL streptomycin. The cells were cultured at 37 °C in a humid-ified atmosphere containing 5% CO2.

Preparation of [3H]-Labeled High Molecular Weight Hyaluronan. [3H]-labeled highmolecular weight hyaluronan ([3H]HA) of>1,000 kDawas prepared according to the methods described by Underhill andToole (1) with some modifications. Confluent Detroit 551 fibro-blasts were incubated with 10 μCi/mL of D-[1,6-3H(N)]glucosaminehydrochloride (American Radiolabeled Chemicals). The condi-tioned medium was pooled, digested with 0.3 mg/mL pronase(Merck), and precipitated with ethanol. The pellets were resus-pended in distilled water, and reprecipitated with 1.5% (wt/vol)cetylpyridinium chloride (CPC) solution containing 0.03 M NaCl.The step was repeated by dissolving in 0.1% CPC solution con-taining 0.4 M NaCl and precipitating with ethanol. The pelletswere suspended in 5 mM phosphate buffer, pH 7.5 and applied toa Sepharose CL-2B column (1 × 60 cm) equilibrated with 0.5 MNaCl in distilled water. The Vo fractions were collected, pre-cipitated with ethanol, and finally dissolved in 5 mM phosphatebuffer, pH 7.5. The specific activity of purified [3H]HA of >1,000kDa was 1.3–2.7 × 105 dpm/μg (2.2–4.5 kBq/μg). This materialwas confirmed to be degraded with Streptomyces hyaluronidase(Seikagaku).

Assay for Cellular [3H]HA Depolymerization. Cellular depolymeri-zation of high-molecular-weight HA was assayed by culturingconfluent cells in medium containing [3H]HA of >1,000 kDa(40,000 dpm/mL) and by applying themedia to a Sepharose CL-2B(GEHealthcare) column (1× 60 cm) equilibrated with 0.5MNaClin distilled water. The flow rate was 0.65 mL/min, and fractions of2.5 mL were collected. The radioactivity of each fraction was mea-sured by a scintillation counter (Aloka; LSC-6100). The columnwas calibrated with fluoresceinamine (FA)-HA species: FA-HAH1 (1,760 kDa), M1 (907 kDa), L1 (197 kDa), S1 (56 kDa), T1(28 kDa), and U1 (9.8 kDa) (peak top kDa), all of which werepurchased from PG Research. For detection of FA, an excitationwavelength of 490 nm and an emission wavelength of 525 nmwere used.

Screening of Stimulators and Suppressors for [3H]HA-DepolymerizingActivity and Microarray Gene Analysis. Detroit 551 skin fibroblaststreated with or without 10 μM histamine, 10 ng/mL TGF-β1,10 ng/mL IL-1α, 10 ng/mL IL-1β, 10 ng/mL TGF-α, 10 ng/mLepidermal growth factor, 10 ng/mL hepatocyte growth factor,10 ng/mL IFN-γ, or 10 ng/mL TNF-α were incubated with [3H]HAfor 48 h. Cellular [3H]HA-depolymerizing activity was assayedas described above and compared between treated and untreatedcells. Because histamine and TGF-β1 up-regulated and down-

regulated [3H]HA-degrading activity, respectively, genes associ-ated with HA degradation were examined by microarray geneexpression analysis. Detroit 551 cells were cultured with or with-out 10 μMhistamine or 10 ng/mL TGF-β1 for 8 and 24 h, and thentotal RNAs isolated using RNeasy (Qiagen) were subjected toAgilent 44K4 chips (Agilent Technologies). Data were analyzedby calculating fold-expression of genes in histamine-stimulated orTGF-β1–stimulated cells against those in untreated cells.

Apparent Km and Vmax for Cellular HA Depolymerization. A mixtureof [3H]HA and nonlabeled high molecular weight HA (humanumbilical cord HA; Seikagaku) was added to Detroit 551 skin fi-broblasts at concentrations of 100, 300, 600, and 1,250 μg/mL, andcultured in the presence or absence of 10 μM histamine for 72 h.Then, conditioned media were harvested and applied to a Se-pharose CL-2B column. HA depolymerization was estimated bymonitoring decreases in the total radioactivity of high molecularweight fractions. Cellular HA depolymerizing activity (μg/105 cells/72 h) was calculated according to the following formula: HAadded to cells (μg) × (total radioactivity of high molecular weightfractions before culture − those after 72-h culture)/total addedradioactivity/number of cultured cells (× 105 cells).

Antibodies. Rat monoclonal antibody against human KIAA1199was developed according to the established protocols (2) using apeptide of CA762RYSPHQDADPLKPRE777, which corresponds tothe amino acid residues Ala762 to Glu777 of KIAA1199 (GenBankaccession no. NM_018689) and has no homology to other proteins.Individual antibody-expressing clones were screened by immuno-blotting for reactivity with recombinant KIAA1199 protein pro-duced in HEK293 cells and by wheat-germ cell-free expressionsystem (Fig. S6A). Anti-human hyaluronidase 2 (HYAL2) poly-clonal antibodies were raised in a rabbit against a peptide with theamino acid sequence of CF78YRDRLGLYPRFDSAGRSV96 (3)and specific antibodies were affinity-purified using the pep-tide. Antibodies against CD44, clathrin heavy chain (CHC),caveolin-1, α-adaptin, and GAPDH were purchased from SantaCruz Biotechnology. Control rat IgG2aκ was purchased fromSouthernBiotech.

Analysis of HA-Specific Depolymerization.Detroit 551 and KIAA1199/HEK239 cells were cultured in medium containing FA-labeledglycosaminoglycans (GAGs): FA-HAs (H1, M1, L1, S1, T1, andU1); chondroitin sulfate A, C, and D (FA-CSA, FA-CSC, and FA-CSD); dermatan sulfate (FA-DS); heparin (FA-Hep); and hep-aran sulfate (FA-HS) (10 μg/mL each). All of these FA-GAGswere purchased from PG Research. After a 72-h incubation pe-riod, the media were harvested and fractionated on a SepharoseCL-6B (GE Healthcare) column (1 × 35 cm) equilibrated withPBS. Fractions of 1.6 mL were collected at 0.4 mL/min and theamounts of FA-GAGs in each fraction were determined by fluo-rescence counting.

Immunoprecipitation. KIAA1199/HEK293 cells were lysed in ice-cold 50 mM Tris•HCl buffer, pH 7.5 containing 1 mMEDTA, 1%TritonX-100, 150mMNaCl, and amixture of proteinase inhibitors(Roche Diagnostics) (lysis buffer). Lysates were cleared by cen-trifugation, and KIAA1199 in the supernatants was immuno-precipitated using Dynal magnetic beads protein G (Invitrogen)covered withmonoclonal antibody against KIAA1199. Rat IgG2aκwas used as a negative control. Beads were washed and then in-cubated with NuPAGE LDS sample-loading buffer (Invitrogen)for 10 min at 70 °C. The samples were electrophoresed on

Yoshida et al. www.pnas.org/cgi/content/short/1215432110 1 of 9

NuPAGE 4–12% Bis•Tris gels (Invitrogen) and analyzed by immu-noblotting using anti-KIAA1199 antibody and anti-CHC antibody.

Immunoblotting. To examine expression of KIAA1199, CD44,HYAL2, CHC, α-adaptin, caveolin-1, and GAPDH proteins, cellsupernatant homogenates were separated by electrophoresis onNuPAGE 4–12% Bis•Tris gels (Invitrogen) and proteins weretransferred onto polyvinylidene difluoride membranes. The mem-branes were reacted with the antibodies specific to KIAA1199,CD44, HYAL2, CHC, α-adaptin, caveolin-1, or GAPDH. Theantibodies against KIAA1199 and HYAL2 were prepared in thepresent study and others were purchased from Santa Cruz Bio-technology. Primary antibodies were detected with horseradishperoxidase-conjugated secondary antibodies as follows: horserad-ish peroxidase-labeled donkey anti-rat IgG antibody for KIAA1199(Jackson ImmunoResearch); goat anti-rabbit IgG antibody forHYAL2, CD44, caveolin-1, and GAPDH (DAKO); rabbit anti-mouse IgG antibody for α-adaptin (DAKO); and rabbit anti-goatIgG antibody for CHC (DAKO). Immunoreactive bands weredetected with chemiluminescence detection by SuperSignal WestPico Chemiluminescent Substrate system (Thermo Scientific).

RT-PCR and Real-Time PCR. Total RNA was isolated using RNeasy(Qiagen) and cDNA synthesis was done using a High CapacitycDNA Archive Kit (Applied Biosystems). RT-PCR of HYAL1,HYAL2, CD44, and housekeeping gene GAPDH was done, andspecific PCR amplification from the target mRNAs was confirmedby sequencing of PCR products using Applied Biosystems 3730xlDNA Analyzer (Life Technologies). PCR amplification was doneafter denaturing at 95 °C for 5 min on a thermal cycler usingGeneAmp PCR system 9700 (Applied Biosystems) by running 30cycles under the following condition: denaturation for 30 s at94 °C; annealing for 30 s at 55 °C; and extension for 60 s at 72 °C.Nucleotide sequences of the primers were as follows: sense primer5′-CAGGCGTGAGCTGGATGGAGA-3′ and antisense primer5′-GTATGTGCAACACCGTGTGGC-3′ for HYAL1; senseprimer 5′-GAGTTCGCAGCACAGCAGTTC-3′ and antisenseprimer 5′-CACCCCAGAGGATGACACCAG-3′ for HYAL2;sense primer 5′-TCCCAGTATGACACATATTGC-3′ and an-tisense primer 5′-CACCTTCTTCGACTGTTGAC-3′ for CD44;and sense primer 5′-ACCACAGTCCATGCCATCAC-3′ and an-tisense primer 5′-TCCACCACCCTGTTGCTGTA-3′ for GAPDH.The expected sizes of the amplified cDNA fragments for HYAL1,HYAL2, CD44, and GAPDH were 400, 446, 549, and 452 bps,respectively. For quantitative analysis of KIAA1199 expression,cDNA was used as template in a TaqMan real-time PCR assay(ABI Prism 7700 Sequence Detection System; Applied Bio-systems) according to the manufacturer’s protocol. The relativequantification value of KIAA1199 was normalized to endogenousGAPDH. The total gene specificity of nucleotide sequences cho-sen for the primers and probe and the absence of DNA poly-morphisms were ascertained by BLASTN and Entrez onWeb sites(www.ncbi.nlm.nih.gov).

RNA Interference. To identify genes involved in HA depolymeriza-tion, knockdown experiments for the 25 candidate genes (Table S1)were done using two, different chemically synthesized 25-nucleotidesiRNAduplexes per gene, obtained from Invitrogen. The sequencesof the siRNA oligonucleotides are as follows: KIAA1199-1, 5′-AA-ACAUUGAAAUAUUCGCCAUGCUC-3′; KIAA1199-2, 5′-UU-GACAAGGAGGCCAAGACAGUGGU-3′; KIAA1706-1, 5′-AUUGAUACCAUCCAUCCUCACUAGG-3′; KIAA1706-2, 5′-AACCGGUGGCCAUCACUGUGAUUCU-3′; GUCY1A3-1,5′-AUAUUCAACAUAGUCAUGAUCCCGC-3′; GUCY1A3-2,5′-AAUGUAGAUCAUUUGGCCUUUGAGG-3′; CD36-1, 5′-UUGUCUUCUGGAUAAGCAGGUCUCC-3′; CD36-2, 5′-AA-GGUUCGAAGAUGGCACCAUUGGG-3′; PRKAR2B-1, 5′-UAAUCCUGGACUCUGCAUCAUCUUC-3′; PRKAR2B-2,

5′-UAUCAUAGUUACCAACACAUCUUCC-3′; TCF21-1, 5′-CAUUCUCGCAGUUGGAGCUCUCCUC-3′; TCF21-2, 5′-UU-GAAGGUCCUCCACAUCGCUGAGG-3′; SIX1-1, 5′-AAGU-CCACCAGACUGGAGGUGAGGG-3′; SIX1-2, 5′-UAAAGC-CAAACGACGGCAGCAUCGA-3′; KBTBD10-1, 5′-UAACAA-UGCUGGAAUGAUUUCUGGG-3′; KBTBD10-2, 5′-AUAUC-UUGCACAUUUCCGUCAUUGA-3′; FGL2-1, 5′-UUUGGAC-UUCUUUGAACACCUCCUC-3′; FGL2-2, 5′-AACAACAGU-CCGUUUCUGCCUGGGU-3′; C10orf10-1, 5′-UUGUCCAGC-ACAGAGGUGGGCUGUG-3′; C10orf10-2, 5′-UAGGUCAGA-GCUCUGCUGCCCAUCC-3′; PARD6A-1, 5′-UUAUUGCGC-UGGUUGGCGGGCUUGA-3′; PARD6A-2, 5′-AGGUCUUC-CCGGCUACUUCAAUGCC-3′; FAM20A-1, 5′-AAGAUGGC-CAUGUCGAUGACAUUGA-3′; FAM20A-2, 5′-UUAUCAUG-CAGCACUGGGAGAGAGG-3′; ARMC4-1, 5′-AAACUUGG-CAACAUUCGCGAUAGUC-3′; ARMC4-2, 5′-UUCGUAUG-ACUCUUACUGCAGCUCC-3′; FGD4-1, 5′-AUUAGUCU-CCUUCAUCUCAUGGUGC-3′; FGD4-2, 5′-AUGUGAUGC-UGCAAAGUUAAGCUCC-3′; TMTC1-1, 5′-UAAUACAGU-GCUCCCAAAGGUGACA-3′; TMTC1-2, 5′-UUUGGUUUC-AGCUGGAGAGCCUUGU-3′; RASL11A-1, 5′-UAGACCAG-CCGUGAAUACAGCUUGC-3′; RASL11A-2, 5′-AUUUGGA-CAGGGAAUCGACGACCUG-3′; C6orf32-1, 5′-AUACCAGGU-GAUUUCCAGGUUCAGU-3′; C6orf32-2, 5′-UUUCAAGGCC-CUGUAGACUUCUUCC-3′; AK024243-1, 5′-UACUCUUGGU-CCCUGAUCAAAGUCC-3′; AK024243-2, 5′-AUCAGUCUGA-GUUAAAGGCCAGCUC-3′; FLJ35700-1, 5′-UUUCCAAGGG-CAGAGCCAGCAUUUG-3′; FLJ35700-2, 5′-UGAGAAGUCUG-UUCUCGUCACUCUG-3′; FLJ36644-1, 5′-UUAACUGGGAC-CACAUCGCCAUUUG-3′; FLJ36644-2, 5′-UUUAUUAUGAC-CUUCCUCCCGCUGC-3′;KLHL23-1, 5′-AAACAUGUAACAC-CAUAGCUCUCCC-3′;KLHL23-2, 5′-UAUAGAUCCACACUG-UGUCAAGAGC-3′; BX350256-1, 5′-UAGGCCUGCAUAAUAC-AGUCCUAUG-3′; BX350256-2, 5′-UAAUGUGGGAUCCUU-AUCAGCAUGC-3′; ENST00000372493-1, 5′-AAUAUAGGCU-ACGCAGUUGUGGAGG-3′; ENST00000372493-2, 5′-UGAUC-CUCCCAGUUUCUUCUUGCUC-3′; THC2404909-1, 5′-UCAU-CUUUCACUGGAGCCCGGAAGC-3′; THC2404909-2, 5′-UUU-CCAAGGAGUCACGUGGUCGCUC-3′; A_32_P171043-1, 5′-AG-AUGUCGGGCAUUAACUCAGAAGG-3′; and A_32_P171043-2,5′-UUUACUUGUUGGGCUAUGAUACUCC-3′.Two different siRNAs forHYAL2, CD44, CHC, caveolin-1, and

α-adaptin, and nonsilencing control siRNAs were also purchasedfrom Invitrogen. The sequences of the siRNAs are as follows:HYAL2-1, 5′-AAUCUUUGAGGUACUGGCAGGUCUC-3′;HYAL2-2, 5′-AUAUUGUGCCUGUUUGACUAUGCGG-3′;CD44-1, 5′-AUUCAAAUCGAUCUGCGCCAGGCUC-3′;CD44-2,5′-AAAUGCACCAUUUCCUGAGACUUGC-3′;CHC-1, 5′-UG-AAGUAUUGACAUCAAAUUUCCGG-3′;CHC-2, 5′-AAAUU-CUUCUAACUCUGCAAGGCGG-3′; Caveolin-1-1, 5′-AUUU-CUUUCUGCAAGUUGAUGCGGA-3′; Caveolin-1-2, 5′-UUU-CCCAACAGCUUCAAAGAGUGGG-3′; α-adaptin-1, 5′-UAUU-CAGCAACUCAUCGGCUUCCGG-3′; and α-adaptin-2, 5′-UGA-ACAGGUAACCUAUUUGCUUCUC-3′. These siRNAs weretransfected into cells using Lipofectamine RNAiMAX (Invitrogen).

Preparations of Plasmids and Transfectants. The chemically syn-thesized and subcloned cDNAsof humanwild-typeKIAA1199 andfourmissensemutations (R187C,R187H,H783R, andV1109I) (4)were obtained from Takara Bio. The authenticity of the cDNAswas verified by sequencing using Applied Biosystems 3730xl DNAAnalyzer (Life Technologies). Plasmids were prepared by insertingthe cDNAs of KIAA1199 (GenBank accession no. NM_018689),HYAL1 (U_96078), and HYAL2 (U_09577) into the expressionvector pcDNA3.1(-) (Invitrogen) according to the manufacturer’sprotocol. Transient transfection was done using LipofectamineLTX (Invitrogen), and transfectants were used for the experimentsat 48 h after transfection. For preparation of stable transfectants

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of KIAA1199 in HEK293 cells (KIAA1199/HEK293 cells),HEK293 cells were transfected with the pcDNA3.1(-) vectors con-taining the KIAA1199 cDNA and stable transfectants were selectedby culturing in medium containing 800 μg/mL of G418 (Sigma). Theexpression and activity of KIAA1199 were monitored by immuno-blotting and by the HA-degrading assay, respectively. Inhibitoryeffects on depolymerization of [3H]HAwere examined by treatmentof KIAA1199/HEK293 cells with 5 μM monensin (MP Biomedi-cals), 10 mM NH4Cl (Wako), 1 nM bafilomycin A1 (Calbiochem),60 μM dynasore (Merck), or 0.3 μM nocodazol (Calbiochem).

Identification of Nonreducing and Reducing Terminal Sugars ofDepolymerized HA. KIAA1199/HEK293 cells were cultured in me-dium containing [3H]HA (1,000,000 dpm/mL) for 72 h, and ra-diolabeled products in the medium were detected on a SepharoseCL-2B column. Depolymerized [3H]HA in the fractions was pre-cipitated with ethanol, dissolved in distilled water, then fraction-ated on a column (1 × 107 cm) of Sephadex G-25 (GEHealthcare)after digestion with β-N-acetylglucosaminidase (Sigma) or se-quential digestion with β-glucuronidase (Sigma) and then β-N-acetylglucosaminidase. The elution position ofN-acetylglucosamine(GLcNA) used as a standard was determined according to previousmethods (5). This method enabled us to identify the nonreducingterminal sugar of the depolymerized [3H]HA, because these en-zymes cleave individual sugar residues from the nonreducing ter-minus. To determine the reducing terminal sugar of depolymerizedHA,KIAA1199/HEK293 cells were cultured for 72 h in themediumcontaining nonlabeled human umbilical cord HA (100 μg/mL). Themedium concentrated with ethanol was applied to a Sepharose CL-2B column. The depolymerized HA in the fractions were collected,precipitated with ethanol, and finally dissolved in distilled water.Pyridylamination of the reducing terminal sugar of depolymerizedHAwas done using 2-aminopyridine according to previous methods(6). Pyridylaminated HA (PA-HA) was acid hydrated with 4 NHCl,followed by N-acetylation with 5% (vol/vol) acetic anhydride (7).PA-HAwas purified by reverse-phase HPLC using a TSK-gel SugarAXI (4.6 mm inner diameter × 15 cm; Tosoh) with a 0.7 M po-tassium borate buffer, pH 9.0 containing 10% (vol/vol) acetonitrileat a flow rate of 0.3mL/min at 65 °C. PA-GLcNAwas detected at anexcitation wavelength of 310 nm using an emission wavelength of380 nm.

Immunofluorescence Microscopy for KIAA1199 in Detroit 551 SkinFibroblasts and KIAA1199/HEK293 Cells. Detroit 551 fibroblasts andKIAA1199/HEK293 cells were grown on glassmultichamber slides(BDBiosciences) to 70–80% confluence, washed in PBS, and fixedwith 4% (wt/vol) paraformaldehyde in PBS for 10 min. Afterwashing in PBS containing 0.05% Tween 20 (PBS-T), the cells werereacted with rat anti-KIAA1199 monoclonal antibody conjugatedto Alexa-Flour 488. The samples were counterstained with TO-PRO-3 (Invitrogen) and mounted in vectashield (Vector). Fordouble-immunostaining of KIAA1199 and CHC in KIAA1199/HEK293 cells, the cells were fixed with 4% (wt/vol) para-formaldehyde, reacted with rat anti-KIAA1199 antibody conju-gated to Alexa-Flour 488 and goat anti-CHC antibody conjugatedto Alexa-Flour 555, and then mounted in vectashield without nu-clear counterstaining. As for controls, samples were reacted withnonimmune IgG conjugated to Alexa-Flours. Conjugation ofAlexa-Flours to antibodies and nonimmune IgG was performed byAlexa-Fluor antibody labeling kit (Invitrogen) according to themanufacturer’s protocol. These samples were observed using ZeissLSM 510 confocal microscope (Carl Zeiss).

Cellular Distribution of Exogenously Added HA in Detroit 551 SkinFibroblasts and KIAA1199/HEK293 Cells. The cells grown on glassmultichamber slides were incubated in the presence or absence of0.1 mg/mL biotin-labeled high molecular weight HA of 1,410 kDa(PG Research) at 37 °C for 1 h. The cells were washed with PBS

and then fixed with 4% (wt/vol) paraformaldehyde in PBS. Afterwashing in PBS-T, incubation with streptavidin conjugated toAlexa-Fluor 488 (Invitrogen), and nuclear counterstaining withTO-PRO-3, they were mounted in vectashield and observed usingZeiss LSM 510 confocal microscope. As for control, the cells wereincubated with biotin-labeled high-molecular-weight HA di-gested with Streptomyces hyaluronidase (Seikagaku), followed bythe immunostaining described above.

Determination of Extracellular, Cell Surface-Associated, and IntracellularHA.KIAA1199/HEK293 and control HEK293 cells were cultured insix-well plates in themedium containing [3H]HA (400,000 dpm/mL)for 1, 6, or 24 h, and washed with ice-cold PBS. The medium andwash were combined and designated as “extracellular” fraction. Thecells were removed from the well by incubation with 0.25% trypsin(Sigma) containing 1mMEDTA for 3min at 37 °C andwashed withPBS. The resulting cell suspension and wash were combined andcentrifuged at 4 °C. The resulting supernatant was designated as“cell surface-associated” fraction. The cell pellet was digested with400 μg/mL proteinase K (Invitrogen) in 50 mM Tris•HCl, pH7.4 at37 °C for 24 h, and digestion product was designated as the “in-tracellular” fraction. The radioactivity of each fraction was mea-sured by a scintillation counter (Aloka; LSC-6100).

Detection of HA-Degrading Activity of HYAL1 by Zymography.KIAA1199/HEK293 cells were transfected with expression vec-tors containing the HYAL1 cDNA or empty vectors (Mock). Thecell lysates were electrophoresed on SDS gels containing 100 μg/mLhuman umbilical cord HA (Seikagaku) under nonreducing con-ditions (8). After electrophoresis, the gels were washed with 3%(vol/vol) Triton X-100 in distilled water at 4 °C for 1 h to removeSDS and then incubated in 100 mM sodium formate buffer, pH 3.7containing 1% Triton X-100 and 150 mMNaCl at 37 °C overnight.Then, the gels were treated with 0.1 mg/mL pronase (Merck) in20mMTris•HCl, pH 8.0 for 2 h at 37 °C. TheHAdegrading activityof HYAL1 was detected as a transparent band against a bluebackground in the gels stained with 0.5%Alcian Blue 8GX (Wako).

Coprecipitation of KIAA1199 Protein with GAGs. KIAA1199 protein-GAG complexes were precipitated by CPC according to previousmethods (9, 10) with somemodifications. KIAA1199/HEK293 cellswere washed three times with 50 mM phosphate buffer, pH 6.0 andhomogenized in 50 mM phosphate buffer, pH 6.0 containinga mixture of proteinase inhibitors (Roche Diagnostics). Aliquots ofthe homogenate supernatant (50 μL) were mixed with 50 μL of1mg/mL aqueous solutions of nonlabeledGAGs: CSA, CSC, CSD,DS, Hep, HS, HA-H2 (1,452 kDa), HA-M2 (1,039 kDa), HA-L2(219 kDa), HA-S2 (52 kDa), and HA-T2 (28 kDa) (PGResearch),or H2O (negative control). After the samples were incubated for1 h at 37 °C, 1% CPC [final concentration; (wt/vol)] was added tothe GAG/lysate mixtures and incubated for 1 h. The precipitateswere pelleted by centrifugation, washed three times with 1 mL of1% CPC containing 30 mM NaCl, and then dissolved in 50 μL ofNuPAGE LDS sample-loading buffer (Invitrogen). The sampleswere electrophoresed on NuPAGE 4–12% Bis•Tris gels (In-vitrogen) under reducing conditions and immunoblotted with anti-KIAA1199 monoclonal antibody.

Binding of KIAA1199 to HA-Sepharose. HA-Sepharose was preparedby incubation of 120 mg human umbilical cord HA (Seikagaku)in 10 mL of water with 10 mL EAH Sepharose 4B beads (GEHealthcare) according to previous methods (11). After addition of0.28 g 1-ethyl-3-(3-dimethylaminopropyl)carbodiiamide hydro-chloride (Thermo Scientific), the pHwasmaintained at 4.5–5.0 for24 h by addition of 0.1 M HCl. The beads were washed sequen-tially with 1 M NaCl, 0.1 M Tris•HCl buffer, pH 8.1, 0.05 M for-mate buffer, pH 3.1, and then equilibrated with 50 mM phosphatebuffer, pH 6.0 containing 0.1% (vol/vol) Triton X-100. KIAA1199/

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HEK293 cells were washed three times with 50 mM phosphatebuffer, pH 6.0 containing 0.1% (vol/vol) Triton X-100 and ho-mogenized in the same buffer containing a mixture of proteinaseinhibitors (Roche Diagnostics). Aliquots of the homogenate su-pernatants were incubated with control unsubstituted or HA-cou-pled Sepharose beads for 1 h at 37 °C. The samples were washedwith equilibration buffer and materials bound to the beads wereeluted by incubation with NuPAGE LDS sample-loading buffer(Invitrogen). Eluates were then electrophoresed on NuPAGE 4–12% Bis•Tris gels (Invitrogen) and immunoblotted with anti-KIAA1199 monoclonal antibody. The specificity of KIAA1199binding was examined by competition experiments in which freeGAGs including HA-H2, CSA, CSC, CSD, DS, Hep, and HS(0.5 mg/mL each) were incubated with homogenate supernatantsfor 1 h at 37 °C before application to HA-Sepharose beads.

HA-Degrading Activity of Recombinant KIAA1199. Wheat-germ cell-free protein synthesis was done usingWEPRO1240HExpression kit(CellFree Sciences) according to the manufacturer’s protocol. HA-degrading activity of recombinant KIAA1199 was measured by in-cubation of the synthesized recombinant protein with [3H]HA(40,000 dpm/mL) in 50 mM acetate buffer, pH 4.0 and pH 5.0 or50 mM phosphate buffer, pH 6.0 and pH 7.0 at 37 °C, followed bySepharose CL-2B column chromatography and scintillation count-ing of the fractions.

Human Tissue Samples.Normal human skin tissues were taken witha biopsy punch from the faces of the three volunteers (women; agerange of 61–65 y) at Stephens & Associates. Collection of skintissues was approved by the Institutional Review Board of Ste-phens & Associates and informed consent was obtained from thevolunteers before surgery. Synovial tissues were obtained at the

time of arthroplasty from knee joints of the patients with OA(n = 10) or RA (n = 8). Noninflammatory synovial tissues (n = 3)were taken at arthroscopic examination approximately 1 y afterreconstruction surgery of the anterior cruciate ligament. OA andRA were diagnosed according to the criteria of the AmericanCollege of Rheumatology (12, 13). Informed consent was ob-tained from the patients for the experimental use of the surgi-cally removed synovial samples according to the ethics guidelinesof Keio University Hospital.

Immunohistochemistry. Immunohistochemical studies were doneon paraffin sections of formalin-fixed human skin and rheumatoidsynovium.After rehydration, antigen retrievalwasdonewith 1mMEDTA, pH8.0 for 15min at 95 °C. Endogenous peroxidase activityand nonspecific binding were blocked by incubation with 3% (vol/vol) H2O2 in PBS for 15 min and 10% (vol/vol) donkey serum inPBS containing 1% BSA and 0.05% Tween 20 (BSA/PBS-T) for30 min, respectively. The sections were incubated with themonoclonal antibody against KIAA1199 or control rat IgG2aκ ata concentration of 4 μg/mL in BSA/PBS-T for 18 h at 4 °C. Afterwashing in PBS, the sections were incubated with horseradishperoxidase-conjugated donkey anti-rat IgG antibody (dilution of1:500 in BSA/PBS-T; Jackson ImmunoResearch) for 1 h at roomtemperature. Color was developed by reacting with the VectorDAB (brown; Vector Laboratories) or Vector VIP kit (purple;Vector Laboratories).

In Situ Hybridization. Digoxigenin-labeled KIAA1199 anti-senseand sense riboprobes were synthesized (GenBank accession no.NM_018689, nucleotides 5729–6352). In situ hybridization wasdone on paraffin sections of formalin-fixed human normal skinand rheumatoid synovium as described previously (14).

1. Underhill CB, Toole BP (1979) Binding of hyaluronate to the surface of cultured cells.J Cell Biol 82(2):475–484.

2. Kishiro Y, Kagawa M, Naito I, Sado Y (1995) A novel method of preparing rat-monoclonal antibody-producing hybridomas by using rat medial iliac lymph node cells.Cell Struct Funct 20(2):151–156.

3. Harada H, Takahashi M (2007) CD44-dependent intracellular and extracellularcatabolism of hyaluronic acid by hyaluronidase-1 and -2. J Biol Chem 282(8):5597–5607.

4. Abe S, Usami S, Nakamura Y (2003) Mutations in the gene encoding KIAA1199protein, an inner-ear protein expressed in Deiters’ cells and the fibrocytes, as thecause of nonsyndromic hearing loss. J Hum Genet 48(11):564–570.

5. Reissig JL, Storminger JL, Leloir LF (1955) A modified colorimetric method for theestimation of N-acetylamino sugars. J Biol Chem 217(2):959–966.

6. Takemoto H, Hase S, Ikenaka T (1985) Microquantitative analysis of neutral andamino sugars as fluorescent pyridylamino derivatives by high-performance liquidchromatography. Anal Biochem 145(2):245–250.

7. Hase S, Tsuji Y, Matsushima Y (1972) Structural studies on a glucose-containing polysac-charide obtained from Micrococcus lysodeikticus cell walls. IV. Isolation of a disaccharide,6-O-(strontium 2-acetamido-2-deoxy-β-D-mannopyranosyluronate)-D-glucose. J Biochem72(6):1549–1555.

8. Guntenhöner MW, Pogrel MA, Stern R (1992) A substrate-gel assay for hyaluronidaseactivity. Matrix 12(5):388–396.

9. Bono P, Rubin K, Higgins JM, Hynes RO (2001) Layilin, a novel integral membraneprotein, is a hyaluronan receptor. Mol Biol Cell 12(4):891–900.

10. Lee TH, Wisniewski HG, Vilcek J (1992) A novel secretory tumor necrosis factor-inducible protein (TSG-6) is a member of the family of hyaluronate binding proteins,closely related to the adhesion receptor CD44. J Cell Biol 116(2):545–557.

11. Armstrong SE, Bell DR (2002) Measurement of high-molecular-weight hyaluronan insolid tissue using agarose gel electrophoresis. Anal Biochem 308(2):255–264.

12. Altman R, et al.; Diagnostic and Therapeutic Criteria Committee of the American Rheu-matism Association (1986) Development of criteria for the classification and reporting ofosteoarthritis. Classification of osteoarthritis of the knee.Arthritis Rheum 29(8):1039–1049.

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14. Sugiyama Y, Shimada A, Sayo T, Sakai S, Inoue S (1998) Putative hyaluronan synthasemRNA are expressed in mouse skin and TGF-β upregulates their expression in culturedhuman skin cells. J Invest Dermatol 110(2):116–121.

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Fig. S1. Endogenous HA-specific depolymerization by Detroit 551 fibroblasts in culture. The cells in 12-well plates were cultured with FA-GAGs: FA-HA H1, FA-CSA, FA-CSC, FA-CSD, FA-DS, FA-Hep, and FA-HS (10 μg/mL each) for 72 h. FA-GAGs in the conditioned media were fractionated on a Sepharose CL-2B column.Dotted lines indicate FA-GAGs without incubation.

Fig. S2. Abrogation of HA depolymerization by siRNA-mediated knockdown of KIAA1199 in normal human skin fibroblasts. KIAA1199 expression wasknocked down by two different siRNAs (KIAA1199-1 or KIAA1199-2) in Detroit 551 (A), HS27 (B), and NHDF-Ad cells (C). The cells were cultured with [3H]HA for48 h, and degraded HA fragments in the media were detected by chromatography on a Sepharose CL-2B column. Dotted lines indicate [3H]HA without in-cubation. The effect of siRNAs on KIAA1199 protein expression was assessed by immunoblotting with anti-KIAA1199 antibody (Insets). Control siRNA, controlnonsilencing siRNA; GAPDH, a loading control.

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Fig. S3. HA-specific depolymerization by KIAA1199 stable transfectants in HEK293 cells (KIAA1199/HEK293 cells). Control HEK293 (○) and KIAA1199/HEK293cells (●) were cultured for 72 h with FA-labeled GAGs: FA-CSA, FA-CSC, FA-CSD, FA-DS, FA-Hep, and FA-HS, and HA species with different molecular weights(FA-HA H1, M1, L1, S1, T1, or U1) (10 μg/mL each). Depolymerization of these GAGs in the media was determined by chromatography on a Sepharose CL-6Bcolumn. Dotted lines indicate GAG without incubation.

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Fig. S4. Effect of HYAL1 or HYAL2 expression on depolymerization of [3H]HA in KIAA1199/HEK293 cells. KIAA1199/HEK293 cells were transfected with emptyvectors (Mock) or vectors containing HYAL1 or HYAL2 cDNA, and the transfectants were incubated with [3H]HA for 6 h. HA degradation was detected by size-exclusion chromatography. Overexpression of HYAL1 and HYAL2 was assessed by HA zymography and immunoblotting, respectively. Expression of KIAA1199protein in the transfectants was confirmed by immunoblotting with anti-KIAA1199 antibody.

Fig. S5. Cellular localization of KIAA1199 and exogenously added HA in Detroit 551 skin fibroblasts and KIAA1199/HEK293 cells. (A and B) Immunohisto-chemistry of KIAA1199 with anti-KIAA1199 antibody (A) or nonimmune IgG (B) in skin fibroblasts. The plasma membrane is depicted by dotted line. Arrows,KIAA1199 positive signals; N, nucleus. (Scale bars, 5 μm.) (C and D) Double immunostaining of KIAA1199 (green) and CHC (red) with anti-KIAA1199 and anti-CHC antibodies (C) or nonimmune IgG (D) in KIAA1199/HEK293 cells. (Inset) High-power view of the boxed area. The plasma and nuclear membranes areshown by dotted lines. Arrows, KIAA1199+ signals; N, nucleus. (Scale bars, 5 μm; Inset, 0.5 μm.) (E and F) HA localization in skin fibroblasts (E) and KIAA1199/HEK293 cells (F) after incubation with biotin-labeled HA, followed by reaction with streptavidin conjugated to Alexa-Fluor 488. Arrows, HA+ structures; N,nucleus. (Scale bars, 5 μm.) (G) HA localization in KIAA1199/HEK293 cells incubated with biotin-labeled HA that had been digested with Streptomyces hyal-uronidase. N, nucleus. (Scale bar, 5 μm.)

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Fig. S6. Preparation of recombinant KIAA1199 protein and its HA depolymerizing activity. (A) Recombinant KIAA1199 protein was expressed in a wheat-germcell-free expression system using the KIAA1199 cDNA. Proteins were separated by NuPAGE using 4–12% Bis•Tris gels, and the gels were stained with Coomassiebrilliant blue (CBB) or subjected to immunoblotting with anti-KIAA1199 antibody (IMB). For controls, a wheat-germ cell-free expression system was carried outin the absence of cDNA. Arrows, KIAA1199 protein. (B) [3H]HA was incubated with synthesized recombinant KIAA1199 protein at pH 4.0, 5.0, 6.0 or 7.0, and theHA depolymerizing activity was examined by column chromatography. Dotted lines indicate [3H]HA without incubation.

Fig. S7. KIAA1199-dependent depolymerization of HA by cultured OA and RA synovial fibroblasts and by cells expressing wild-type or mutant KIAA1199proteins reported in hearing loss patients. (A) Expression of KIAA1199 protein in OA and RA synovial fibroblasts was knocked down with two different siRNAsfor KIAA199 (KIAA1199-1 or KIAA1199-2), and then the cells were incubated with [3H]HA for 48 h. [3H]HA degrading activity in the media was determined bysize-exclusion chromatography. Dotted lines indicate [3H]HA without incubation. KIAA1199 protein expression was assessed by immunoblotting with anti-KIAA1199 antibody (Insets). The data obtained by transfection with one KIAA1199 siRNA (KIAA1199-1) are shown. Control siRNA, control nonsilencing siRNA;GAPDH, a loading control. (B) HEK293 cells were transfected with vectors containing cDNAs of wild-type (WT) KIAA1199 or four missense mutations (R187C,R187H, H783R, and V1109I), and incubated with [3H]HA for 24 h. HA depolymerization in the media was determined as described above. Expression of the wild-type and mutant forms of KIAA1199 was confirmed by immunoblotting with anti-KIAA1199 antibody (IMB).

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Table S1. Genes up-regulated by histamine and down-regulated by TGF-β1 in Detroit 551 skin fibroblasts

Gene name Accession no.

Fold-induction (control: 1.0)

8 h 24 h

Histamine TGF-β1 Histamine TGF-β1

KIAA1199 NM_018689 5.4 0.1 3.1 0.1KIAA1706 NM_030636 4.5 0.3 2.6 0.2GUCY1A3 NM_000856 2.1 0.3 3.6 0.2CD36 NM_001001547 2.6 0.3 2.5 0.2PRKAR2B NM_002736 2.6 0.3 2.5 0.1TCF21 NM_003206 4.7 0.5 3.0 0.3SIX1 NM_005982 2.2 0.3 2.1 0.3KBTBD10 NM_006063 3.8 0.4 4.5 0.3FGL2 NM_006682 9.0 0.4 9.1 0.2C10orf10 NM_007021 11.4 0.5 8.7 0.4PARD6A NM_016948 2.7 0.5 2.1 0.2FAM20A NM_017565 3.0 0.3 2.2 0.3ARMC4 NM_018076 2.5 0.2 2.6 0.4FGD4 NM_139241 5.6 0.1 3.9 0.1TMTC1 NM_175861 2.1 0.3 2.4 0.5RASL11A NM_206827 5.2 0.3 3.2 0.4C6orf32 AB002384 17.5 0.5 3.7 0.3AK024243 AK024243 2.5 0.4 2.2 0.2FLJ35700 AK093019 4.6 0.5 2.7 0.3FLJ36644 AK093963 4.9 0.5 2.8 0.1KLHL23 BC016950 2.0 0.3 2.6 0.3BX350256 BX350256 3.6 0.3 2.7 0.2ENST00000372493 ENST00000372493 2.0 0.4 4.2 0.5THC2404909 THC2404909 2.1 0.4 2.9 0.4A_32_P171043 A_32_P171043 3.4 0.1 2.1 0.0

Detroit 551 cells were cultured in the presence and absence of 10 μM histamine or 10 ng/mL TGF-β1. At 8 h and 24 h after thetreatment, total RNAs were isolated and subjected to microarray gene-expression analysis. Values (fold-induction) represent the ratioof expression levels in the stimulated cells compared with the untreated control cells.

Table S2. Distribution of [3H]HA in extracellular, cell surface and intracellular compartments after addition toKIAA1199/HEK293 and control HEK293 cells

Fraction

1 h 6 h 24 h

KIAA1199/HEK293 HEK293 KIAA1199/HEK293 HEK293 KIAA1199/HEK293 HEK293

Extracellular 99.75 ± 0.17 99.29 ± 0.97 99.64 ± 0.35 98.66 ± 1.62 99.61 ± 1.47 98.31 ± 1.31Cell surface 0.19 ± 0.01 0.65 ± 0.01 0.21 ± 0.01 1.17 ± 0.08 0.16 ± 0.01 1.37 ± 0.01Intracellular 0.06 ± 0.01 0.06 ± 0.01 0.15 ± 0.01 0.17 ± 0.02 0.23 ± 0.01 0.32 ± 0.01

[3H]HA was incubated with KIAA1199/HEK293 and control HEK293 cells for 1, 6, or 24 h, and radioactivity in the extracellular, cellsurface, and intracellular fractions was counted as described in SI Materials and Methods. Recovery is expressed as a percentage of thetotal radioactivity. Values represent mean ± SD (n = 3).

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