Cathepsin L increased level upon Ras mutants expression:the role of p38 and p44/42 MAPK signaling pathways

Lorena Urbanelli • Francesco Trivelli • Luisa Ercolani •

Eleonora Sementino • Alessandro Magini • Brunella Tancini •

Raffaella Franceschini • Carla Emiliani

Received: 12 January 2010 / Accepted: 12 May 2010 / Published online: 4 June 2010

� Springer Science+Business Media, LLC. 2010

Abstract The involvement of Ras and three major Ras

effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral

guanine nucleotide exchange factor in the regulation of

lysosomal proteases cathepsin L and B in human fibroblasts

was compared. We found that cathepsin L cell content was

increased by active Ras overexpression through Raf- and

PI3K-mediated signaling pathways, while cathepsin B pro-

cessing was altered by active Ras overexpression. Cathepsin

L increased level following active Ras overexpression cor-

relates with an increase of p38 MAPK activation and content

and with an increase of p44/42 MAPK activation, so we

investigated the role of these signaling pathways using

pharmacological inhibitors. Unexpectedly, the p38 MAPK

inhibitor SB203580 produced an increase of cathepsin L

content, while the p44/42 MAPK signaling cascade inhibitor

U0126 produced a remarkable shift of cathepsin L pro-

cessing in favor of procathepsin L. In both cases, cathepsin B

level and processing were not affected. The analysis of

CTSL1 gene transcript demonstrated that cathepsin L pro-

tein and transcript correlate both in fibroblasts expressing

Ras mutants and in pharmacologically treated cells, thus

indicating a transcriptional up-regulation.

Keywords Cathepsin L � Cathepsin B � Ras �p44/42 MAPK � p38 MAPK

Introduction

Cysteine cathepsins belong to the papain-like family (pep-

tidase C1A) sharing similar amino acid sequences and fold

[1, 2]. There are 11 human cysteine cathepsins and among

them, cathepsins L and B are the most abundant. However,

they differ in specificity. Their activities are regulated by

endogenous protein inhibitors, cystatins [3]. Among these

proteases, cathepsin L shows the strongest affinity to col-

lagen I and IV, elastin and fibronectin [4], whereas

cathepsin B degrades laminin, fibronectin and collagens

[5, 6]. Both cathepsin L and B are widely expressed in human

cells and overexpressed in tumors [7, 8]. Overexpression and

secretion of procathepsin L in human melanoma cells

increase their tumorigenicity and switch their phenotype

from non-metastatic to highly metastatic [9, 10]. In addition,

downregulation of cathepsin L expression by antisense

cDNA significantly impaired malignancy property of gli-

oma, myeloma and osteosarcoma cells [11–13]. Lysosomal

cathepsin L processes intracellular proteins, while secreted

procathepsin L regulates function of extracellular proteins,

for instance the third component of complement [14] and

interleukin-8 converting enzyme [15], and may increase

tumor cell invasiveness by mediating degradation of extra-

cellular matrix (ECM) components [12, 13, 16].

Cathepsin B was the first lysosomal protease to be

associated with breast carcinoma [17]. Subsequently,

increased expression and changes in the localization of this

L. Urbanelli � F. Trivelli � L. Ercolani � E. Sementino �A. Magini � B. Tancini � R. Franceschini � C. Emiliani (&)

Department of Experimental Medicine and Biochemical

Sciences, University of Perugia, Via del Giochetto, 06123

Perugia, Italy

e-mail: emiliani@unipg.it

Present Address:E. Sementino

Cancer Genetics and Signaling Program, Fox Chase Cancer

Center, Philadelphia, PA, USA

R. Franceschini

Universita degli Studi ‘‘Guglielmo Marconi’’, Rome, Italy

123

Mol Cell Biochem (2010) 343:49–57

DOI 10.1007/s11010-010-0497-3

enzyme have been observed in many different tumors [18],

and suggested to be a prognostic marker in patients with

breast, lung, colon and ovarian carcinomas, as well as

gliomas and melanomas [8, 19, 20]. In tumor cells,

cathepsin B can be secreted into the extracellular envi-

ronment or be associated with the cell surface [21]. As in

the case of cathepsin L, the secreted cathepsin B can

activate other proteases acting downstream in the catalytic

cascade or directly degrade the ECM proteins [22].

Ras is a member of the small GTPase superfamily of

proteins that function as molecular switches to transmit

inside the cell extracellular signals. Ras is frequently

mutated in many different human cancers [23] and the role

Ras plays in the transformed cell phenotype has been

extensively studied [24]. Ras exerts its effect through

activation of a diverse spectrum of downstream effector-

mediated cytoplasmic signaling pathways [25, 26]. The

most studied of these Ras effector pathways are the Raf/

MEK/ERK mitogen-activated protein kinase signaling

cascade, the phosphatidylinositol 3-kinases (PI3Ks) [27]

and the guanine nucleotide exchange factor (GEF) for the

Ral small GTPase [28, 29]. There is evidence that the role

of specific effectors in mediating Ras transformation is

dependent on cell type and species variations [30].

A frequent result of Ras activation is the increased

expression of lysosomal enzymes [31] and ectopic

expression of activated Ras has been reported to cause up-

regulation of lysosomal cathepsins in a variety of cultured

cells, including mouse fibroblasts [32], mouse melanocytes

[33], rat fibroblast and epithelial cells [34]. How secreted

lysosomal proteases are activated and remain stable at the

near neutral pH of the extracellular environment is not fully

understood, but roles for these proteases in cleavage events

outside the cell are expected, as cathepsins L, B and D can

degrade ECM proteins in vitro [25].

In this study, we evaluated cathepsin L and B content

following active Ras mutants expression in human fibro-

blasts. Effector domain mutants of Ras, specifically acti-

vating Raf, PI3K or Ral guanine nucleotide exchange factor

(RalGEF), and pharmacologic inhibitors of p38 and p44/42

MAPK were utilized to determine the role of these path-

ways in mediating lysosomal cysteine proteases cell content

regulation. We observed that expression of RasV12, double

mutants RasV12S35 and RasV12C40, or pharmacological

inhibition of p38 and p44/42 MAPK signaling cascades

resulted in an increase of cathepsin L but not of cathepsin B

level, while inhibition of p44/42 MAPK signaling cascade

also affected cathepsin L but not cathepsin B processing. In

addition, we evaluated cathepsin L transcript and demon-

strated that cathepsin L protein content correlates with

cathepsin L gene transcript, both in fibroblasts infected with

Ras mutants and in pharmacologically treated cells, indi-

cating a transcriptional up-regulation.

Materials and methods

Cell lines

Human diploid fibroblasts were obtained from skin biopsy

of a healthy individual. Cells were cultured in Dulbecco’s

modified Eagle’s medium (DMEM) containing 10% (v/v)

heat-inactivated fetal bovine serum (FBS, Biokrom, Berlin,

Germany), 2 mM glutamine, 10 U/ml penicillin, 10 lg/ml

streptomycin. The viability of the cells was estimated by

examining their ability to exclude trypan blue (0.1% in

0.9% NaCl). U0126 and SB203580 were dissolved in

DMSO at 10 and 25 mM concentration, respectively.

All chemicals were added at 80% confluence, after 12 h

incubation with 0.1% FBS. Experiments were conducted

24–48 h after chemicals addition. DMSO was added as

vehicle control in all experiments and the total DMSO

concentration was \0.1% in both treated and non-treated

samples.

pBABE vectors and Ras mutants expression

Expression vectors encoding effector domain mutants of

activated H-RasV12 have been described previously: the

amino acid substitutions shown by the GTPases render them

constitutively GTP bound and thus activated, while effector

domain mutants of H-RasV12 impair them in specific

effector interactions. Briefly, the E37G mutant retains

binding to RalGEFs but is reduced in its ability to bind Raf or

PI3K. Similarly, the Y40C mutant retains PI3K binding but

is reduced in Raf and RalGEF binding, while the T35S

mutant retains Raf binding but is reduced in PI3K and Ral-

GEF binding [35]. Vectors expressing RasV12 and mutants

RasV12C40, RasV12G37 and RasV12S35 were obtained by

subcloning Ras expressing segments from pSG5 vector into

the EcoRI site of pBABE-PURO retroviral expression vec-

tor. Fibroblasts were infected with a pBABE-PURO retro-

virus encoding for RasV12, RasV12C40, RasV12G37,

RasV12S35 or with pBABE-PURO vector as control. All

retroviruses were produced by transfecting 10 lg of each

plasmid by CaCl2 method into the Phoenix packaging cell

line. Infected fibroblasts were then cultured for 7 days under

puromycin selection (1 lg/ml), as previously described

[36]. pBABE-PURO and pSG5-RasV12, RasV12C40,

RasV12G37 and RasV12S35 were kindly provided by

Dr. Giuliana Pelicci, Department of Experimental Oncology,

European Institute of Oncology, Milan, Italy.

Cell extracts

Cell samples were washed twice with 0.9% NaCl (5009g

for 10 min in top centrifuge) and suspended in 10 mM

sodium phosphate pH 6.0 buffer and 0.1% (v/v) Nonidet

50 Mol Cell Biochem (2010) 343:49–57

123

P40 detergent. After 1 h incubation on ice, they were

harvested, sonicated using a 100 W ultrasonic disintegrator

(Virtis, Gardiner, NY, USA) equipped with a microtip at

12 mW wave amplitude (one sonication, 15 s), then cen-

trifuged at 16,0009g for 20 min. All procedures were

carried out at 4�C. The protein content was determined by

the method of Bradford using BSA as a standard [37].

Immunoblotting

Cell proteins (20–30 lg) were electrophoresed on acryl-

amide gel at 150 V for 1 h. Proteins were transferred to

nitrocellulose membrane at 100 V for 1 h. The membrane

was blocked by 5% non-fat dry milk in Tris-buffered saline

with 0.1% Tween 20 (TBST) at room temperature for 1 h.

Goat polyclonal anti-cathepsin L and rabbit polyclonal

anti-cathepsin B were from Santa Cruz Biotechnology

(Santa Cruz, CA, USA). Rabbit polyclonal anti-P-p38

MAPK, anti-p38 MAPK, anti-P-p44/42 MAPK and anti-

p44/42 MAPK antibodies as well as goat anti-rabbit HRP-

linked secondary antibody were from Cell Signaling

Technology (Beverly, MA, USA). As internal control, the

membrane was also probed 1 h at room temperature with

mouse monoclonal anti-b-tubulin (Sigma, St. Louis, MO,

USA) or goat polyclonal anti b-actin (Santa Cruz) anti-

bodies. Goat anti-mouse HRP-linked and goat anti-rabbit

HRP-linked secondary antibodies were used according to

manufacturer. Immunoblots were detected by chemilumi-

nescence using either Lumi-GLO chemiluminescent

reagent (Cell Signaling Technology) or ECL (GE Biosci-

ences). Densitometric evaluation of bands was obtained

with Image J software [38].

Quantitative PCR

RNA was extracted using Trizol reagent (Sigma) according

to the manufacturer’s instructions. 1 lg of RNA was

reverse-transcribed into cDNA using random hexamers and

SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad,

CA, USA). cDNA was used as template for the estimation

of CTSL1 gene expression by quantitative PCR (Q-PCR) in

a Stratagene Mx3000P Q-PCR machine (Agilent Tech-

nologies, La Jolla, CA, USA). Reactions were performed in

triplicate using Brilliant� II SYBR� Green Q-PCR Master

Mix (Agilent Technologies). Sequences used for the

amplification of CTSL1 gene were 50-CGG CAT GAA

TGAAGA AGG AT (forward) and 50-TGG GCT TAC

GGT TTT GAA AG (reverse). b-Actin (ACTB) gene was

amplified as endogenous control using the following

primers 50-AGA AAA TCT GGC ACC ACA CC (forward)

and 50-GGG GTG TTG AAG GTC TCA AA (reverse).

Data were analyzed using the DDCt method. DCt was

calculated subtracting the average Ct value of ACTB to the

average Ct value of CTSL1 gene for each sample. DDCt is

the difference between the DCt for each samples and the

DCt of pBABE infected or untreated fibroblasts as control.

The reported fold expression, expressed as RQ (relative

quantity), was calculated by 2-DDCt.

Results

Ras mutants expression in human fibroblasts

To investigate if Ras is an upstream modulator of cathep-

sins L and B in human fibroblasts, we induced the over-

expression of an active form of Ras by infecting them with

a retrovirus expressing a Ras mutant that is constitutively

activated (RasV12), in comparison with the retroviral

vector alone as control. Equal amounts of total cellular

protein were resolved by SDS-PAGE and cathepsins L and

B were visualized by immunoblotting. In human fibro-

blasts, cathepsin L is synthesized as a proenzyme, then is

proteolytically processed to generate a single chain form of

the enzyme. Single chain cathepsin L is subsequently

cleaved into a heavy chain and a light chain linked by a

disulfide bond [39]. By immunoblotting, we detected pro-

cathepsin L and single chain, finding a remarkable increase

of the enzyme level in RasV12 expressing fibroblasts, as

compared to cathepsin L content in control cells infected

with the empty vector (Fig. 1).

We determined which Ras effector pathways were

responsible for the increased cathepsin L content. For these

analyses, we utilized cells expressing one of three Ras

double mutants (RasV12S35, RasV12G37, RasV12C40)

[35]. These mutants are constitutively active and possess an

additional mutation impairing their interaction with

downstream Ras effectors. Nevertheless, they retain spe-

cific activating properties: the RasV12S35 mutant binds

and activates Raf but is reduced in PI3K and RalGEF

binding, the RasV12G37 mutant binds and activates Ral-

GEF but is reduced in its ability to bind to Raf or PI3K and

similarly, the RasV12C40 mutant binds and activates PI3K

but is reduced in Raf and RalGEF binding. We observed

that the level of cathepsin L in RasV12S35 and

RasV12C40 expressing cells was decreased as compared to

cathepsin L content in RasV12 expressing cells, but it was

still higher than in control cells infected by the vector

alone, as it can be visualized by densitometric analysis

(Fig. 1c). In RasV12G37 expressing cells, the cathepsin L

level was decreased as compared to RasV12, and also to

double mutants RasV12S35 and RasV12C40, and it was

equivalent to that of control cells infected with empty

vector. Ras double mutant results show that Ras-mediated

increase of cathepsin L content in human fibroblasts is

mediated by both Raf and PI3K signaling pathways.

Mol Cell Biochem (2010) 343:49–57 51

123

In human fibroblasts, Ras activation negatively affects

the expression of the lysosomal aspartic protease cathepsin

D [40], so Ras cannot be at the basis of a general up-

regulation of cathepsins. To investigate if Ras activation

was able to affect the expression of another lysosomal

cysteine protease or was specific for cathepsin L, we per-

formed immunoblotting with antibodies against cathepsin

B (Fig. 1d). Similar to cathepsin L and other lysosomal

proteases, cathepsin B is initially synthesized as a proen-

zyme, which is proteolytically processed to generate an

active single chain protease, then the single chain protease

is further cleaved into a heavy chain and a light chain. By

immunoblotting, we detected procathepsin B, single and

heavy chain forms of the enzyme, but we did not observe a

clear increase of cathepsin B cell content upon Ras

expression. However, a clear change in the relative quan-

tities of the different biosynthetic forms of cathepsin B was

evident. A shift from the accumulation of heavy chain to

the accumulation of single chain form was clearly detected

upon RasV12 expression. The level of cathepsin B in

RasV12C40 and RasV12G37 expressing cells was com-

parable to those of control cells infected by the vector

ras

P-p44/p42

p44/p42

β-actin

p38

P-p38

proCatL

(a) (c)

(d)

(e)

(f)

0.0

0.3

0.6

0.9

P-

scCatL

proCatB

scCatB

hcCatB

0.0

3.0

6.0

9.0

12.0

15.0(b)

Rel

ativ

e Q

uant

ity

Fig. 1 Analysis of cathepsin L, cathepsin B, p44/42 MAPK and p38

MAPK activation in fibroblasts infected with Ras mutants. aFibroblast extracts infected with the retroviral constructs pBABE-

RasV12, pBABE-RasV12C40, pBABE-RasV12G37, pBABE-

RasV12S35 or with the retrovirus alone were sized by SDS-PAGE,

probed with an anti-Ras antibody, stripped and re-probed with anti-

cathepsin L, anti-cathepsin B, anti-P-p44/42 MAPK, anti-p44/42

MAPK, anti-P-p38 MAPK and anti-p38 MAPK antibodies. As

internal control, the membrane was also probed with an anti-b-actin

antibody. b Gene expression analysis by real time Q-PCR. Reactions

were performed in triplicate, using SYBR green binding to detect

amplification. ACTB gene was used as endogenous control. The fold

expression of CTSL1 gene in fibroblasts infected by Ras mutants,

with respect to those infected with the vector alone as control, is

represented. The value is expressed as relative quantity. The

average ± SD of three different experiments is reported. c Densito-

metric analysis of results reported in a for anti-cathepsin L. dDensitometric analysis of results reported in a for anti-cathepsin B. eDensitometric analysis of results reported in a for anti-P-p44/42

MAPK and anti-p44/42 MAPK. f Densitometric analysis of results

reported in a for anti-P-p38 MAPK and anti-p38 MAPK. Barsrepresent the ratio between the intensity of the band signals and

b-actin. Each densitometric measure is the average ± SD of

three independent analyses. sc single chain, hc heavy chain

52 Mol Cell Biochem (2010) 343:49–57

123

alone. In RasV12S35 expressing cells, the cathepsin B cell

content was slightly increased as compared to that of

control cells infected with empty vector, but no change in

cathepsin B processing was observed, suggesting that a

slight Ras-mediated increase in cathepsin B expression in

human fibroblasts could be mediated by Raf signaling

pathway, but the alteration of cathepsin B processing is

presumably due to additional effector pathways.

Ras may mediate its action through the activation of

multiple downstream signaling cascades. We previously

reported that cathepsin D expression correlates more

accurately with p38 MAPK than with p44/42 MAPK

activation [40], so we investigated both p38 MAPK and

p44/42 MAPK activation and content upon Ras mutants

expression. We found that RasV12 expression induced

activation of p44/42 MAPK without an increase of its level

(Fig. 1e), and a remarkable increase of both p38 MAPK

activation and level (Fig. 1f). On the other hand,

RasV12C40 and RasV12S35 mutants did not increase p44/

42 MAPK activation and p38 MAPK activation and/or

content with respect to control cells infected with the

vector alone, while RasV12G37 mutant induced a decrease

of p44/42 MAPK activation, p38 MAPK activation and

p38 MAPK content, compared to the control. These results

suggested that p44/42 MAPK and p38 MAPK activation is

concomitant with an increase of cathepsin L cell content,

while a reduction of p44/42 MAPK activation, and p38

MAPK activation and p38 MAPK level are associated with

a decrease of cathepsin L content, as shown by the

RasV12G37 mutant analysis.

The high level of cathepsin L content upon RasV12

expression suggested us to investigate the expression of

cathepsin L at mRNA level, to assess if the molecular

mechanism underlying this increase was a transcriptional

up-regulation. We analyzed cathepsin L gene expression by

performing real time Q-PCR (Fig. 1b). RasV12 expressing

cells displayed a 12-fold increase of cathepsin L transcript

in comparison with fibroblasts infected with the vector

alone as control. In addition, cells infected with double

mutants RasV12S35 and RasV12C40 also showed a 2-fold

increase of cathepsin L transcript, thus showing a corre-

lation between cathepsin L protein and transcript.

Effect of p38 MAPK inhibitor SB203580 and p44/42

MAPK inhibitor U0126 on cathepsin L and B level

We further investigated the role of the activation of p38

MAPK and p44/p42 MAPK on the expression of cathep-

sins L and B using pharmacological inhibitors. The role of

p38 MAPK as modulator of cathepsin L was investigated

using SB203580 [41]. Immunoblotting results are reported

in Fig. 2a, and the corresponding densitometric analyses

are reported in Fig. 2c–f. We treated human fibroblasts for

a 24 h incubation time and observed an increased content

of p44/p42 MAPK, probably as a consequence of an

induction of Raf-1 activation, that was previously reported

[42], without an increased activation of p44/p42 MAPK. In

addition, we observed that a 24 h treatment does not

influence p38 MAPK activation and content. However, in

this condition, cathepsin L content unexpectedly increased

in a dose-dependent manner, and in particular the increase

was more evident for the procathepsin L form of the

enzyme. At the same time, cathepsin B level and pro-

cessing remained similar to the untreated control, although

a slight decrease of the heavy chain form could be observed

at 25 lM concentration. To investigate the molecular basis

underlying the increase of cathepsin L protein in

SB203580-treated cells, CTSL1 gene expression was

assessed by real time Q-PCR (Fig. 2b). SB203580-treated

cells showed a dose-dependent increase of cathepsin L

transcript in comparison with untreated cells, demonstrat-

ing a correlation between cathepsin L protein and transcript

levels.

To modulate p44/42 MAPK signaling, we incubated

human fibroblasts with increasing concentrations of U0126

as inhibitor [43] for a prolonged incubation time (24 h) and

the results obtained are reported in Fig. 3a, while the cor-

responding densitometric analysis is reported in Fig. 3c–f.

Immunoblottings of p44/p42 MAPK and phospho-p44/p42

MAPK showed that after a 24 h treatment no decrease of

the expression and activation of p44/p42 MAPK could be

detected in fibroblast extracts. However, the treatment

induced a clear increase of p38 MAPK activation at 30 lM

concentration. Cathepsin L analysis showed an increase of

the enzyme and a concomitant change in the relative

quantities of its different biosynthetic forms, as a shift from

the accumulation of single chain to the accumulation of

procathepsin L was clearly detectable. The analysis of

cathepsin B expression showed no decrease of enzyme

content and no significative differences in terms of post-

translational processing, thus underlying that the effect is

specific for cathepsin L. We determined the amount of

cathepsin L transcript in U0126-treated cells by real time

Q-PCR (Fig. 3b), and observed a dose-dependent increase

of cathepsin L transcript, in comparison with untreated

cells as control. This result provides evidence that U0126

treatment induces an increase of cathepsin L expression.

Discussion

Cathepsin L and B oncogenic properties are generally

thought to involve their activity outside the cells once they

have been secreted. It is conceivable that they could aid

metastasis by participating in the degradation of the ECM

(through which metastasizing cells must migrate) or

Mol Cell Biochem (2010) 343:49–57 53

123

activating other proteases [1, 44, 45]. However, the exact

mechanism by which these lysosomal enzymes aid tumor

progression remains unclear. Recent findings have sug-

gested that an alternative localization of cathepsin L may

also account for its tumorigenic potential: cathepsin L

isoforms that are devoid of a signal peptide were shown to

be present in the nucleus where they cleave the CDP/Cux

transcription factor [46, 47].

Here, we provide evidence that overexpression of a

constitutively active Ras in human fibroblasts remarkably

increases cathepsin L but not cathepsin B cell content. The

increase in cathepsin L level was mediated by the activa-

tion of Raf and PI3K pathways downstream of Ras, as

demonstrated using cells expressing double mutants

RasV12S35 and RasV12C40. When these results were

compared to those reported for rodent cells [34], specific

differences were observed. In human fibroblasts from skin

biopsy, the increase in cathepsin L content upon RasV12

overexpression is mediated by RasV12S35 and

RasV12C40 mutants, but the increase of cathepsin L cell

content upon double mutants RasV12S35 and RasV12C40

expression is six times lower in comparison with that upon

RasV12 expression. On the other hand, in immortalized rat

fibroblasts, the increase of cathepsin L cell content is

mediated almost exclusively by the RasV12S35 mutant, as

cathepsin L levels upon RasV12S35 expression were

comparable to those upon RasV12 expression. This indi-

cated that a single effector pathway is mainly responsible

p44/p42

β-actin

p38

P-p44/p42

P-p38

0 5 10 25 µM SB203580

0 µM 5 µM 10 µM 25 µM

(a)(c)

(d)

(e)

(f)

proCatL

scCatL

proCatB

scCatB

hcCatB

(b)

0.0

2.0

4.0

6.0

8.0

0 µM 5 µM 10 µM 25 µM

Rel

ativ

e Q

uant

ity

Fig. 2 Analysis of cathepsin L, cathepsin B, p44/42 MAPK and p38

MAPK activation in fibroblasts treated with SB203580. a SB203580

was added to fibroblasts at 70–80% confluence after 12 h incubation

with 0.1% FBS. Cells were lysed 24 h after exposure, then extracts

were sized by SDS-PAGE and blotted. The membrane was probed

with an anti-cathepsin L antibody, stripped and re-probed with

cathepsin B, anti-P-p44/42 MAPK, anti-p44/42 MAPK, anti-P-p38

MAPK and anti-p38 MAPK antibodies. As internal control,

the membrane was also probed with an anti-b-actin antibody.

A representative experiment is reported and very similar results were

obtained after a 48 h treatment. b Gene expression analysis by real

time Q-PCR. Reactions were performed in triplicate, using SYBR

green binding to detect amplification. ACTB gene was used as

endogenous control. The fold expression of CTSL1 gene in

SB203580-treated fibroblasts with respect to untreated cells is

represented. The value is expressed as relative quantity. The

average ± SD of three different experiments is reported. See Fig. 1

for c–f description. sc single chain, hc heavy chain

54 Mol Cell Biochem (2010) 343:49–57

123

for Ras-mediated cathepsin L increase in rodent fibroblasts,

while other signaling pathways in addition to Raf and PI3K

cascades are presumably involved in the increase of

cathepsin L expression mediated by active Ras in human

fibroblasts. The analysis of cathepsin L transcript in Ras

expressing fibroblasts provided evidence that the increase

of cathepsin L protein cell content was due to higher levels

of CTSL1 gene transcript, suggesting that cathepsin L

transcription is up-regulated by Ras activation, although a

diminished rate of cathepsin L transcript degradation could

not be excluded. The regulation of cathepsin L expression

has been extensively investigated [48–50] and regulatory

sites essential for cathepsin L promoter activity were

identified in a 50-bp region, containing one CCAAT motif

and two GC boxes, on which bound NF-Y and Sp1/Sp3

transcription factors, respectively. However, further studies

would be necessary to identify the Ras responsive elements

in CTSL1 gene promoter.

Cathepsin L increased cell content following active Ras

expression appears to be concomitant with an increase of

p44/42 MAPK activation and p38 MAPK activation and

content. However, the use of a p38 MAPK pharmacolog-

ical inhibitor provided evidence that when p38 MAPK is

inhibited the expression of cathepsin L increases in a dose-

dependent manner, without a concomitant increase of p44/

42 MAPK activation or p38 MAPK content or activation.

On the other hand, when p44/42 MAPK activation is

pharmacologically inhibited, cathepsin L content clearly

shifts toward an higher content of the proenzyme form,

without any increase of p44/42 MAPK activation or p38

p44/p42

β-actin

p38

P-p44/p42

P-p38

0 5 10 30 µM U0126

(a)(c)

(d)

(f)

(e)

0 µM 5 µM 10 µM 30 µM

proCatL

scCatL

proCatB

scCatB

hcCatB

0.0

2.0

4.0

6.0

0 3µM 10µM 30µM

(b)

Rel

ativ

e Q

uant

ity

Fig. 3 Analysis of cathepsin L, cathepsin B, p44/42 MAPK and p38

MAPK activation in fibroblasts treated with U0126. a U0126 was

added to fibroblasts at 70–80% confluence after 12 h incubation with

0.1% FBS. Cells were lysed 24 h after exposure, then extracts were

sized by SDS-PAGE and blotted as described. The membrane was

probed with an anti-cathepsin L antibody, stripped and re-probed with

cathepsin B, anti-p44/42 MAPK, anti-P-p44/42 MAPK, anti-p38

MAPK and anti-P-p38 MAPK antibodies. As internal control, the

membrane was also probed with an anti-b-actin. A representative

experiment is reported and very similar results were obtained after a

48 h treatment. b Gene expression analysis by real time Q-PCR.

Reactions were performed in triplicate, using SYBR green binding to

detect amplification. ACTB gene was used as endogenous control.

The fold expression of CTSL1 gene in U0126-treated fibroblasts with

respect to untreated cells is represented. The value is expressed as

relative quantity. The average ± SD of three different experiments is

reported. See Fig. 1 for c–f description. sc single chain, hc heavy

chain

Mol Cell Biochem (2010) 343:49–57 55

123

MAPK level, but with a parallel increase of p38 MAPK

activation. This finding implies that the inhibition of p44/

42 MAPK pathway triggers, concomitantly with p38

MAPK activation, post-translational mechanism affecting

the maturation of cathepsin L. It was reported that upon its

biosynthesis, cathepsin L can be targeted to small dense

core vesicles and to the dense cores of multivesicular

bodies, as well as to lysosomes and to the plasma mem-

brane for selective secretion [51]. However, U0126 treat-

ment appears to block at least a key step in these passages,

but further studies are needed to understand where is pro-

cathepsin L in the cell, stable and avoiding proteolytic

activation. To evaluate if the increase of cathepsin L con-

tent in SB203580- and U0126-treated cells was due to an

inhibition of procathepsin L secretion or to a change at the

mRNA level, we analyzed the level of CTSL1 transcript.

We demonstrated that increased levels of cathepsin L

protein were due to a higher amount of CTSL1 gene

transcript. As the level of secreted cathepsin L was not

evaluated, it is possible that high levels of transcript could

also produce an increase of enzyme secretion. Overall

results show that in our condition of pharmacological

treatment, no decrease of cathepsin L content could be

observed by incubation with p44/42 and p38 MAPK

inhibitors, as it could be expected by cathepsin L increased

cell content upon active Ras expression. Our results sug-

gest that a complex interplay between the p44/42 and p38

MAPK signaling cascades regulates cathepsin L expression

and the modulation of a single cascade leads to the unex-

pected result of either an increase of cathepsin L expression

and/or a change of its post-translational processing.

The modulation of cathepsin L cell content by Ras

mutants and inhibitors of p38 and p44/42 MAPK pathways

is clearly specific for this protease and not for another

lysosomal cysteine protease, cathepsin B. Despite the fact

that both lysosomal enzymes are considered a hallmark of

tumor cells, here we show that they respond differently not

only to Ras oncogene activation, but also to the modulation

of p38 MAPK and p44/42 MAPK signaling pathways. The

cascade of p44/42 MAPK is activated by many growth

factors that are involved in the development of tumors,

such as those of the EGF family [52], while p38 MAPK is

activated by many stress stimuli, such as inflammatory

cytokines, UV irradiation and oxidative stress [53]. Our

results suggest that cathepsin L, and not cathepsin B, is the

first lysosomal cysteine protease to be altered when p38

MAPK and p44/42 MAPK signaling pathways are modu-

lated in human fibroblasts, by either Ras oncogene or other

stressing stimuli. Besides, recent findings about the

increased expression of nuclear cathepsin L in cancer

suggest further investigations about the localization of

cathepsin L upon modulation of its cell content by different

treatments.

Acknowledgements This work was supported by COFIN-PRIN,

FIRB from ‘‘Ministero dell’Istruzione, Universita e Ricerca’’ (Italy)

and ‘‘Fondazione Cassa di Risparmio di Perugia’’ (2008.021.375)

grants to C.E. Authors disclose any actual or potential conflicts of

interest including any financial, personal or other relationships with

other people or organizations within 3 years of beginning the work

submitted that could inappropriately influence their work.

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