Panx1 Regulates Cellular Properties of Keratinocytesand Dermal Fibroblasts in Skin Development andWound HealingSilvia Penuela1, John J. Kelly1, Jared M. Churko1,3, Kevin J. Barr2, Amy C. Berger2 and Dale W. Laird1,2

Pannexin1 (Panx1), a channel-forming glycoprotein is expressed in neonatal but not in aged mouse skin.Histological staining of Panx1 knockout (KO) mouse skin revealed a reduction in epidermal and dermal thicknessand an increase in hypodermal adipose tissue. Following dorsal skin punch biopsies, mutant mice exhibited asignificant delay in wound healing. Scratch wound and proliferation assays revealed that cultured keratinocytesfrom KO mice were more migratory, whereas dermal fibroblasts were more proliferative compared with controls.In addition, collagen gels populated with fibroblasts from KO mice exhibited significantly reduced contraction,comparable to WT fibroblasts treated with the Panx1 blocker, probenecid. KO fibroblasts did not increase a-smooth muscle actin expression in response to TGF-b, as is the case for differentiating WT myofibroblasts duringwound contraction. We conclude that Panx1 controls cellular properties of keratinocytes and dermal fibroblastsduring early stages of skin development and modulates wound repair upon injury.

Journal of Investigative Dermatology (2014) 134, 2026–2035; doi:10.1038/jid.2014.86; published online 27 March 2014

INTRODUCTIONThe channel-forming family of glycoproteins, known aspannexins, consists of three members: Panx1, Panx2, andPanx3 (Panchin et al., 2000). Although all three pannexinsform functional single-membrane channels (Penuela et al.,2009), Panx1, which is the most ubiquitously expressed of thethree (Baranova et al., 2004), has been more extensivelystudied (Penuela et al., 2013). The role of Panx1 as amechanosensitive ATP-release channel (Bao et al., 2004) hasbeen linked to paracrine signaling involving Ca2þ wavepropagation (Locovei et al., 2006), vasoconstriction (Billaudet al., 2011), airway defense (Seminario-Vidal et al., 2011),and even facilitating HIV viral infection (Seror et al., 2011).Panx1 has also been shown to mediate cellular responses inthe immune system, acting as the ATP-release channel of‘find-me’ signals from apoptotic cells to monocytes duringtheir recruitment for cell clearance (Chekeni et al., 2010).Under pathological conditions, Panx1 has been reported toincrease the severity of seizures in status epilepticus (Santiago

et al., 2011). Panx1 channel opening increases neuronal celldeath under ischemic conditions (Thompson et al., 2006,2008), and promotes death of enteric neurons in colitis(Gulbransen et al., 2012).

In normal development, all three pannexins have beenreported to have an important role in different cell types.Panx1 is important in differentiation of keratinocytes(Celetti et al., 2010), Panx3 in osteoblasts (Bond et al., 2011;Ishikawa et al., 2011) and chondrocytes (Iwamoto et al., 2010;Bond et al., 2011), and Panx2 in development of neuronalcells (Swayne et al., 2010). Panx1 has been implicated in thedifferentiation of cardiac fibroblasts into myofibroblasts inresponse to ATP released from cardiomyocytes (Dolmatovaet al., 2012). Recently, it was also reported that mast cell–derived histamine can activate ATP release from humansubcutaneous fibroblasts via Panx1 channels through H1receptor activation (Pinheiro et al., 2013). Interestingly,Panx1 is not only functional at the cell surface but has alsobeen shown to be expressed in intracellular compartments,such as the endoplasmic reticulum, where it is proposed tofunction as an intracellular Ca2þ leak channel (VandenAbeele et al., 2006; D’Hondt et al., 2011).

In adult human skin, we have previously reported theexpression of Panx1 and Panx3 in the different layers andappendages of the skin with various localization profiles(Penuela et al., 2007; Cowan et al., 2012). In mouse skin,we detected Panx1 and Panx3 expression as early as dayE13.5 in the developing epidermis and dermis (Celetti et al.,2010). At the cellular level, we demonstrated that anoverexpression of Panx1 in rat epidermal keratinocytes(REKs) reduced their proliferation rate, and disrupted theirnormal differentiation in a model of organotypic epidermis

ORIGINAL ARTICLE

1Department of Anatomy and Cell Biology, University of Western Ontario,London, Ontario, Canada and 2Physiology and Pharmacology, University ofWestern Ontario, London, Ontario, Canada

Correspondence: Silvia Penuela, Department of Anatomy and Cell Biology,University of Western Ontario, Dental Science Building DSB 00076, London,N6A5C1, Ontario, Canada. E-mail: spenuela@uwo.ca

3Current address: Department of Medicine (Cardiology), Stanford School ofMedicine, Stanford, California 94305, USA.

Received 16 October 2013; revised 8 January 2014; accepted 2 February2014; accepted article preview online 12 February 2014; published online27 March 2014

Abbreviations: KO, knockout; REK, rat epidermal keratinocyte; a-SMA,a-smooth muscle actin; WT, wild type

2026 Journal of Investigative Dermatology (2014), Volume 134 & 2014 The Society for Investigative Dermatology

grown in three-dimensional cultures from these engineeredREKs (Celetti et al., 2010). In addition, Panx1 expression in basaland squamous skin cell carcinomas, as well as in the moredeadly melanomas, has been shown to change with malignanttransformation of the skin. In human keratinocyte tumors, bothPanx1 and Panx3 are significantly reduced (Cowan et al., 2012),whereas mouse melanomas express high levels of Panx1 thatcan be downregulated to revert them into a more normalmelanocytic phenotype (Penuela et al., 2012).

Using the Panx1 knockout (KO) mouse model reportedby Qu et al. (2011), we set out to evaluate the role of Panx1in skin development and response to injury in vivo.We discovered that Panx1 is necessary for early skindevelopment, has decreasing expression in aged skin, and isupregulated again upon wounding. Panx1 has a role in themigration of keratinocytes and regulates differentiation ofdermal fibroblasts into myofibroblasts for proper woundcontraction. When Panx1 is missing, fibroblasts proliferatemore and do not properly differentiate in response to TGF-b,leading to the formation of fibrotic scars.

RESULTSPanx1 expression decreases in aging dorsal skin

Levels of endogenous Panx1 protein expression in the dorsalskin of neonatal mice and in mice up to 4 weeks of age werehigher than those in adult mice (12 weeks old) and in agedmice of up to 18 months (Figure 1a). This same trend wasobserved in studies using Panx1 immunofluorescence labelingof skin sections of different ages (Figure 1b and c). Panx1 islocalized at the cell surface of basal keratinocytes and hairfollicles of neonatal skin (Figure 1b, white arrowheads),whereas dermal fibroblasts and differentiated keratinocytesof the upper layers of the epidermis present an intracellulardistribution (Figure 1b, stars and open arrowheads). As theskin ages, Panx1 not only decreases in the different layers ofthe skin, but its localization pattern is less prevalent at the cellsurface of the different skin cell types (Figure 1c).

Characterization of the Panx1 KO mouse model

As previously described and characterized by Qu et al. (2011),a gene-targeting strategy was used to generate Panx1-nullmice that were confirmed to lack Panx1 in tissues in whichexpression is normally abundant, such as the brain, thymus,and spleen (Figure 1d and e). Neonatal dorsal skin of thePanx1 KO was also devoid of Panx1. To verify the absence ofany Panx1 transcript in the KO mouse, brain and thymustissue from WT and KO mice were used for qPCR of Panx1(Figure 1e). Panx1 is absent in KO mice dorsal skin (Figure 1f),whereas Panx3 was found to be upregulated in young andadult Panx1 KO skin compared with controls (Figure 1g).

Decreased dermal thickness and increased hypodermal fat inPanx1 KO mice

Panx1 KO mice are viable, fertile, and do not show an overtskin phenotype. However, comparing histological sections ofdorsal skin from 1-day to 7-week-old WT mice with thosefrom KO mice, we detected a significant decrease in totaldermal area (dermis and epidermis) of KO dorsal skin in every

age group. At the same time, the hypodermal fat area wassignificantly increased in the KO mice as early as 4 days afterbirth and this trend prevailed into adulthood (Figure 2).

Panx1 is upregulated in dorsal skin wounds and its depletiondelays wound healing, increasing fibrosis

Healing of dorsal wounds on 12-week-old males was sig-nificantly reduced in KO mice compared with that in WTcontrols at every stage of healing, including inflammation,proliferation, and tissue remodeling (Figure 3a). Althoughwounds eventually closed, there was a prominent scar onthe KO mouse skin, which when examined in histologicalsections revealed an area of increased fibrosis (Figure 3b) thatwas quantified by measuring the maximum wound depth(N¼ 4, n¼ 12, Po0.01).

As mentioned before, mouse dorsal skin at 12 weeks of agehas low basal levels of Panx1 expression (Figure 1c). How-ever, upon wounding, Panx1 expression detected by immu-nohistochemistry is increased near the site of the wound asearly as 3 days after surgery in WT dorsal skin (Figure 3c).Panx1 is apparent in both dermis and epidermis, reachingmaximum expression at day 6 after surgery. By day 13, Panx1protein expression returns to basal levels around the woundsite, but it is still highly expressed in the new re-epithelializedtissue (above dotted wound line, Figure 3c).

Primary basal keratinocytes express high levels of Panx1 in earlystages of differentiation

To elucidate the mechanism responsible for delayed woundhealing in KO mice skin, we isolated primary keratinocytesfrom neonatal dorsal skin. WT basal keratinocytes areenriched in Panx1 (Figure 4a). A growth curve assay showedno significant differences in the growth rate of KO keratino-cytes compared with that in WT controls (Figure 4b). How-ever, an in vitro scratch wound assay revealed a significantlydysregulated migration/proliferation of KO keratinocytes after12 hours of migration in the presence of serum (Figure 4c). Totest for the ability of keratinocytes to differentiate in culture,cells were exposed for 2 days to medium containing CaCl2solution. Panx1 protein levels were significantly reduced 2days after calcium treatment (Figure 4d). However, both KOand WT keratinocytes could start to differentiate in vitro,as the differentiation marker involucrin was significantlyincreased in both groups of keratinocytes (Figure 4e). Therewere no apparent differences between WT and KO cells in thelevels of CK14, a marker of basal keratinocytes (Figure 4e).

To test whether the Panx1 present in these primary keratino-cytes was forming active channels at the cell surface, weperformed a sulforhodamine B dye uptake assay as describedearlier by our group (Celetti et al., 2010). WT keratinocytescould readily uptake dye, whereas KO cells exhibitedsignificantly reduced dye uptake. Dextran-rhodamine, whichis too large to pass through pannexin channels, did not enterthe cells, indicating that the cells were healthy and the plasmamembrane was intact (Figure 4f). All dye uptake assays wereperformed in the presence of physiological levels of calciumand magnesium to minimize any potential uptake due tocalcium-gated connexin hemichannels.

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Panx1 KO–derived primary dermal fibroblasts proliferate lessthan WT fibroblasts and have reduced dye uptake capabilities

Primary dermal fibroblasts from WT neonatal mice abundantlyexpress Panx1 (Figure 5a). KO-derived fibroblasts grew fasterthan WT control fibroblasts as revealed by growth curveanalysis (Figure 5b). This difference in growth was due toproliferation and not due to decreased apoptosis, as shown bydifferential labeling of nuclei with the proliferation markerphospho-histone 3 (Figure 5c) and by apoptotic nuclei labeledwith Apoptag (TUNEL assay, Figure 5d). There was no

significant difference between WT and KO fibroblasts in thetotal migrated distance after a scratch wound assay in vitro(Figure 5e).

Similar to primary keratinocytes, WT primary dermalfibroblasts were also capable of sulforhodamine B dye uptake(Figure 5f), indicating that there were functional channelspresent at the cell surface. Panx1 KO fibroblasts showedsignificantly reduced dye uptake, and dextran rhodaminecontrols were again negative (not shown). The Panx1 blockerprobenecid (PBN) had a dose-dependent response in the

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Figure 1. Panx1 decreases in aging skin and is absent in Panx1 knockout (KO)–derived tissues. (a) Western blots and (b, c) immunohistochemistry revealed that

Panx1 is elevated in neonatal skin and it decreases with aging. Cell surface localization of Panx1 in the basal epidermal keratinocytes and hair follicle

cells (solid arrows) is prevalent in 1-day-old skin (b) but is reduced 4 days after birth (c) and in differentiated keratinocytes (*). (b) Panx1 is also expressed in

dermal fibroblasts (open arrows) of neonatal skin. (d) Western blots of protein lysates from Panx1 KO tissues, revealed no expression of Panx1 compared with

wild type (WT). (e) qPCR of transcripts from brain and thymus confirmed the absence of Panx1 mRNA in the KO mice. (f) Panx1 is absent in dorsal skin

of KO mice. (g) The B43-kDa isoform of Panx3 is highly upregulated in 4-week-old Panx1 KO mouse skin and, to a lesser extent, in adult skin, whereas

the B70-kDa band of Panx3 does not change. Student’s t-test, N¼4, Po0.0001 or Po0.05. Bars ¼ 20mm. Blue, nuclei. Protein sizes in kDa. Gly0, 1, 2 represent

Panx1 bands with different levels of glycosylation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control.

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inhibition of dye uptake by WT fibroblasts but had no addedblocking effect on dye uptake from KO cells (Figure 5f and g).

Contractile properties of Panx1 KO–derived dermal fibroblastsare reduced, and they do not increase smooth muscle actin inresponse to TGF-b stimulation

To test whether Panx1 KO–derived dermal fibroblasts wereable to differentiate into myofibroblasts and efficiently con-tract a free-floating collagen gel, we seeded equal numbers ofWT and KO fibroblasts into collagen lattices. The gel surfacearea was measured for 96 hours. A significant decrease incontraction was observed with KO-derived fibroblasts at alltime points (Figure 6a). This decrease was comparable to thatobserved with WT-derived fibroblasts and subjected to thePanx1 blocker PBN (Figure 6b). A dose-dependent responsewas evident with 0.5 or 1 mM PBN, indicating that the channelblocker had an effect on the contractile properties of thePanx1-rich WT fibroblasts. Control gels without cells orwithout serum showed no significant contraction as expected,suggesting that the contraction is a mechanistic effect of themyofibroblasts (Figure 6b).

Mouse dermal fibroblasts tend to spontaneously differentiatewhen plated on hard surfaces or on collagen-coated plasticdishware (Elliott et al., 2012). Both WT and KO dermalfibroblasts had some level of differentiation and expressedbasal levels of smooth muscle actin (a-SMA) as seen on bothwestern blots and immunofluorescence (Figure 6c and d).However, WT fibroblasts responded to the addition of TGF-bby increasing the levels of a-SMA, a necessary step in normal

differentiation into contractile myofibroblasts. On the otherhand, Panx1 KO fibroblasts failed to further respond to TGF-bmaintaining the same levels of a-SMA, consistent with theirinability to fully contract (Figure 6a). Morphologically, KOfibroblasts did not respond to TGF-b stimulation, whereas WTfibroblasts acquired a contractile myofibroblast morphologyafter stimulation (Figure 6d).

DISCUSSIONNormal skin development requires a carefully orchestrated setof events involving different cell types derived from theectoderm and the mesenchyme. We have previously demon-strated that Panx1 is expressed as early as day E13.5 in thedeveloping epidermis and dermis (Celetti et al., 2010). Inneonatal skin, Panx1 is highly expressed in keratinocytes,fibroblasts, and hair follicle cells. In basal keratinocytes andhair follicles, Panx1 is expressed at the cell surface in a profilereminiscent of previously reported ectopic expression inreference cells (Boassa et al., 2007; Penuela et al., 2007). Askeratinocytes differentiate and move upwards in the epidermallayer, their Panx1 expression decreases and localizes tointracellular compartments. On the basis of our previouswork (Penuela et al., 2009) and other reports (Boassa et al.,2007), we would predict that the most complex glycosylatedform of Panx1 (Gly2) would be more prevalent at the cellsurface, whereas Gly1 and Gly0 would be more abundantintracellularly. The change in Panx1 expression wasrecapitulated in vitro when Panx1-rich primary basalkeratinocytes were induced to differentiate in the presence

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Figure 2. Skin thickness is decreased in Panx1 knockout (KO) mice, whereas hypodermal fat increases compared with wild-type (WT) controls. Histological

analysis of dorsal skin sections from KO mice showed a reduction in dermal area (dermis and epidermis) when compared with WT controls. N¼4, Po0.05. As

early as 4 days after birth, Panx1 KO mouse skin shows an accumulation of hypodermal adipose tissue that persists into adulthood, N¼ 4, Po0.05 (NS, not

significant).

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of calcium, and began to decrease their Panx1 expressionwhile increasing expression of involucrin, a differentiationmarker. However, Panx1 KO–derived primary keratinocytes

were still able to differentiate, so Panx1 may not be requiredfor initiating this process. Panx1 is important for proper skinarchitecture, as Panx1 KO skin tends to have a thinner dermalarea than WT mice. This may be due to the effect of Panx1deletion not only on the keratinocytes but also on the dermalfibroblasts that appear to show dysregulated proliferation anddifferentiation. As skin ages, Panx1 expression decreases in alllayers of the skin, indicating that this channel protein isneeded early on in development but is downregulated as skinmatures. This is similar to earlier reports of Panx1 expressed athigh levels in the embryonic and young neonatal rat brain thatsignificantly decline in adult rat brain (Vogt et al., 2005). Onlywhen the skin is challenged with an injury is Panx1 againneeded at the wound site, with expression peaking around day6 after surgery but returning to basal expression levels typicalof adult mouse skin once the wound is healed.

The thinner dermal area seen on KO mouse skin appears tobe offset by an increase in the hypodermal fat area, whichmay simply occur as a response to the need to maintain athermal balance of the thinner skin. Alternatively, the increasein hypodermal fat could be due to potential changes inhormone levels as has been reported for male mice withlow testosterone (Azzi et al., 2005), or other possible changesin adipocyte regulation that move beyond the scope of thisstudy.

Similarly, without challenge, KO mice of different agesseem to have no overt skin phenotypes other than the changesin thickness seen under histological observations. This may beexplained in part by the upregulation and potential compen-sation of Panx3 in KO skin of Panx1-null mice, particularly atearly stages when Panx1 would be needed for proper devel-opment. However, upon injury of the dorsal skin at 12 weeksof age, the lack of Panx1, even in the presence of Panx3expression in the skin, results in significant delays in woundhealing, and after wound closure the scars that remain on theKO skin are highly fibrotic. We observed a significantdifference between WT and KO skin in all phases of woundhealing. Early stages of wound healing could involve pannex-ins due to a compromised inflammation response at days 1–3.Panx1 has been reported to have a role in the inflammasome(Silverman et al., 2009), is known to be expressed inmacrophages (Pelegrin and Surprenant, 2006), and couldvery likely be involved in the signaling process of immunecells for phagocytic clearance of apoptotic cells (Chekeniet al., 2010). The granulation or proliferation phase is alsosignificantly delayed in which both Panx1-rich keratinocytesand dermal fibroblasts have a major role (Gurtner et al., 2008).Here, the deletion of Panx1 dysregulates migration ofkeratinocytes and increases proliferation of dermalfibroblasts, and these effects may be due in part to a lack ofATP release via Panx1 channels present in these two celltypes. ATP released from Panx1 channels could potentially acton purinergic receptors to activate downstream signaling thataffects proliferation, differentiation, and apoptosis of differentskin cell types, as reviewed by Burnstock et al. (2012). ATPreleased in response to mechanical stimulation and celldamage is involved in proper wound healing (Wang et al.,1990), hence we could speculate that the complex of Panx1

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Figure 3. Panx1 is transiently upregulated at wound sites and its depletion

increases fibrosis and delays wound healing. (a) Wound-healing assays

performed with dermal punch biopsies on the dorsal area of 12-week-old male

mice showed significant delays in wound closure of Panx1-null skin compared

with wild-type controls during all phases of wound repair. Duplicate wounds,

n¼ 28, N¼ 14, Po0.01 or 0.001. Bars indicate SEM. (b) Histology of healed

wounds at 13 days after surgery show significant fibrosis and increased

scar depth in knockout (KO) wounds, whereas controls healed normally.

(c) Immunolabeling of Panx1 (red) at different time points during healing of

wild-type (WT) skin wounds revealed an upregulation of Panx1 at the wound

site (basal levels are low in 12-week-old skin, day 1) as early as 3 days after

surgery, with a peak intensity at day 6. Panx1 expression returns to basal levels

near healed wounds (day 13) but is still expressed in re-epithelialized wound

tissue. Dashed line indicates wound edge. Nuclei, blue. Bars ¼ 20mm.

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and purinergic receptors orchestrates the proper signalingcascade needed for the healing response.

In addition, KO fibroblasts proliferate more in vitro, and canmigrate as well as the WT fibroblasts, but we hypothesize thatonce at the site of injury, they cannot fully respond to TGF-b forproper differentiation into a-SMA-enriched myofibroblasts thatserve to contract the wound (Desmouliere et al., 1993; Tomaseket al., 2002). This lack of differentiation could be due to theabsence of ATP release needed for the proper differentiation and

contraction of fibroblasts (Ehrlich et al., 1986; Dolmatova et al.,2012) or due to other molecules that may pass through thechannel. Some Panx1 channel-independent functions are alsopossible, such as direct binding of the C-tail of Panx1 to actin(Bhalla-Gehi et al., 2010). However, as PBN, although animperfect channel blocker (Silverman et al., 2008), seems toblock the dye uptake function in WT fibroblasts and mimics thecontraction impairment exhibited by KO fibroblasts, the channelfunction of Panx1 is likely involved. The last phase of wound

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Figure 4. Primary keratinocytes derived from knockout (KO) neonatal skin are more migratory than those from wild-type (WT) controls, and their dye uptake

ability is impaired. (a) Panx1 is highly expressed in WT basal keratinocytes, as seen on western blots of total protein lysates, but its depletion does not

significantly alter their growth (b). P40.05, N¼ 6. (c) Scratch wound experiments revealed that Panx1-null primary keratinocytes were more migratory than

controls. N¼ 5, Po0.01. (d) Western blots of differentiating primary keratinocytes from wild-type mice in the presence of 1.4 mM CaCl2 revealed a progressive

decrease (from day 0 to day 2) in the levels of Panx1. N¼ 6, Po0.01. (e) A marker of keratinocyte differentiation (involucrin) increased, whereas basal

marker cytokeratin 14 (CK14) remained unchanged with no significant differences between WT and KO genotypes. (f) Sulforhodamine B dye uptake in Panx1 null–

derived primary cells (KO) was reduced under conditions of physiological calcium and mechanical stimulation when compared with wild-type controls (WT).

Po0.05, N¼ 4. Negative control experiments using dextran-rhodamine revealed no dye uptake. Error bars indicate SEM. GAPDH and b-tubulin were used as

loading controls.

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healing, known as tissue remodeling (Singer and Clark, 1999)may also be compromised in the absence of Panx1 as KO scarsshow increased fibrosis. Fibrotic and hypertrophic scars arecharacterized by the excessive production of collagen and otherextracellular matrix components by the proliferating fibroblaststhat are not correctly remodeled, as is the case of keloid scars(Tredget et al., 1997). Fibrotic scars could be explained byincreased proliferation of the KO fibroblasts at the wound site,but also may be due to defects in extracellular matrixremodeling and collagen reabsorption (Gurtner et al., 2008).

Collectively, we have shown that Panx1 has a keyrole in proper skin development and its expression is notneeded in aging skin, unless there is an injury that requires itsupregulation. In keratinocytes and dermal fibroblasts,Panx1 is required for proper migration and differentiation asKO fibroblasts do not respond to TGF-b by increasinga-SMA to form fully differentiated myofibroblasts thatcan contract properly and aid wound closure. Panx1 maybe a new target for wound-healing treatments andre-epithelialization.

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Figure 5. Dermal fibroblasts from knockout (KO) mice dorsal skin proliferate more than those from wild-type (WT) controls and have impaired dye

uptake function. (a) Dermal fibroblasts from WT neonatal skin are rich in Panx1 as shown by western blot. b-Tubulin was used as loading control. (b) Dermal

fibroblasts from KO neonatal mice subjected to growth curve analysis had a significant increase in proliferation. N¼ 6, Po0.05 or 0.01. (c) Increased cell

proliferation was confirmed by quantification of nuclei, stained with phospho-histone 3 (pHis3), N¼ 3, Po0.001. (d) Apoptag labeling (TdT terminal

deoxynucleotidyl transferase) of nuclei undergoing apoptosis showed no difference between WT and KO primary fibroblasts. (e) Scratch wound experiments

revealed that Panx1-null primary fibroblasts had no significant difference in migration compared with WT controls. N¼5, P40.05. (f) Sulforhodamine-B dye

uptake in Panx1 KO–derived primary cells was reduced under conditions of physiological calcium and mechanical stimulation when compared with WT controls.

WT fibroblasts responded in a dose-dependent manner to probenecid (PBN) inhibition of dye uptake, whereas the Panx1 blocker had no further inhibitory effect on

dye uptake in KO-derived cells. Po0.05, N¼ 4. Error bars indicate SEM (NS, not significant).

S Penuela et al.Panx1 in Skin and Wound Healing

2032 Journal of Investigative Dermatology (2014), Volume 134

MATERIALS AND METHODSDetailed methods in Supplementary Section online.

Ethic statement and animals

Animal experiments were approved by the Animal Use Subcommittee

of the University Council on Animal Care at Western University.

Panx1 KO mice were a kind gift from Dr Vishva Dixit at Genentech

(San Francisco, CA) and were described by Qu et al. (2011). WT mice

of the C57BL/6N strain were from Charles River Canada (Saint-

Constant, PQ).

Western blots

Western blots of tissues and primary cell lysates were performed as

described in the study by Penuela et al. (2007, 2009). Panx1 and

Panx3 antibodies used (specific to the C terminus) were described in

the study by Penuela et al. (2009).

Immunolabeling

Coverslips with primary cells were fixed in either methanol acetone

or 10% neutral buffered formalin and immunolabeled with primary

antibodies as described previously (Penuela et al., 2007). For

proliferation assays, cells were labeled with the M-phase-specific

marker phospho-histone 3 (Millipore, Billerica, MA). For apoptosis

assays, cells were labeled following the Apoptag Fluorescein kit

instructions (Millipore) and nuclei were stained with Hoechst 33342.

Immunolabeling of dorsal skin samples was performed as described

in the study by Celetti et al. (2010). For histology assays,

parallel tissue sections were stained with hematoxylin/eosin. Images

0 24 48 72 96

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Time (hours)

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48- α-Smooth muscle actin

β-Tubulin

WT WT KOTGFβ: ++

WT WT KO KO

*NS

TGFβ: +–+–

WT

KO

No FBS

Control

0.5 mM PROB

1 mM PROB

No cells

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KO

No cells

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KO 2

No FBS

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KO

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***

Figure 6. Contractile properties of dermal fibroblasts are compromised upon Panx1 deletion. (a) In a collagen lattice contractility assay, knockout (KO) dermal

fibroblasts embedded in free-floating collagen gels contracted significantly less than those embedded with wild-type (WT) fibroblasts (two-way ANOVA, N¼ 6, n¼36,

Po0.001) over 4 days. Gels without cells did not contract. (b) Images in a and b are representative of gels at the 96hours time point. WT fibroblasts in a similar time

course contractility assay exposed to 0.5 or 1.0mM of the Panx1 blocker probenecid (PROB) had reduced contractility in a dose-dependent manner (two-way ANOVA,

N¼ 6, n¼ 36, Po0.001 ). Gels without cells or with cells but no fetal bovine serum (FBS) had no significant gel contraction. Bars: SEM. (c) Fibroblasts derived from KO

mice neonatal skin do not respond to the addition of TGF-b1 (NS, not significant), whereas WT fibroblasts respond by increasing the expression of a-smooth muscle actin

(SMA; two-way ANOVA, N¼ 3, Po0.05). In two-dimensional culture, WT fibroblasts in the presence of TGF-b1 can change morphology by differentiating into

contractile myofibroblasts, whereas KO fibroblasts do not change cell morphology (d). SMA, red. Nuclei, blue. Bars ¼ 20mm. b-Tubulin was used as a loading control.

S Penuela et al.Panx1 in Skin and Wound Healing

www.jidonline.org 2033

were collected using a Leica DM IRE2 inverted epifluorescence

microscope as described in the study by Churko et al. (2011a).

ImageJ was used to determine the dermal area (epidermis and dermis)

as well as the hypodermal area (area between dermis and panniculus

carnosus). All statistical analyses were performed using GraphPad

v.4.1.

Primary keratinocyte and dermal fibroblast cultures

Neonatal dorsal skin was dissected from KO and WT pups. After

disinfection, the skin was processed either for primary keratinocyte

extraction and differentiation following the protocol described in the

study by Churko et al. (2012), or for extraction of dermal fibroblasts

and TGF-b differentiation as reported by Churko et al. (2011b).

Quantitative PCR

Total RNA was extracted with Qiagen RNeasy kits (Qiagen, Toronto,

ON, Canada) from brain and thymus dissected from 7-week-old

females from WT and Panx1 KO genotypes. Using the RevertAid H

minus, first-strand cDNA synthesis kit from Fermentas (Burlington,

ON, Canada), total RNA was used to generate the cDNA template.

Panx1 transcript levels were determined using mouse Panx1-specific

primers (forward: 50-ACAGGCTGCCTTTGTGGATTCA-30 and

reverse: 50-GGGCAGGTACAGGAGTATG-30) for qPCR amplification

with iQ SYBR green Supermix (Bio-Rad, Mississauga, ON, Canada) in

a Bio-Rad CFX96 real-time system. Results were normalized to b-2

microglobulin expression.

Cell growth and migration assays

Primary cells were evaluated in a growth curve assay as described

before (Churko et al., 2011b, 2012). Scrape wounding migration

assays were performed as described by Churko et al. (2011b).

Dye uptake assays

Primary cells were subjected to dye uptake assays under physiological

calcium as reported by Celetti et al., (2010) and Penuela et al. (2012).

PBN (Sigma, Oakville, ON, Canada) inhibition of dye uptake was

done at 0.2 mM or 1.0 mM PBN, with pre-incubation at 37 1C in

growth media for 1 hour. All dye uptake solutions and washes

contained the same dose of PBN. Controls were performed using

the vehicle solvent (D-PBS with calcium and magnesium, pH 7.4,

Invitrogen, Burlington, ON, Canada).

Wound healing

Twelve-week-old male mice (WT and KO) were anesthetized with

isoflurane gas and dorsal skin incisions were performed and evaluated

as described by Churko et al. (2011b).

Collagen gel contractility assay

Free-floating collagen gels were prepared from primary mouse

fibroblasts isolated from six wild-type mice and six Panx1 KO mice.

This technique has been described elsewhere (Grinnell et al., 1999;

Ngo et al., 2006; Kelly et al., 2012). For drug-treated gels, the

pannexin-1 inhibitor probenecid (Silverman et al., 2008) was added

to the cell suspensions and culture media to give a final concentration

of 0.5–1 mM.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSWe thank Vishva Dixit (Genentech) for generously providing the Panx1 KOmice. We also thank Daniella Ostojic and Cindy Sawyez for their valuablecontributions to this work. This work was supported by the Canadian Institutesof Health Research (CIHR).

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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