©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell-specific Induction of Distinct Oncogenes of the Jun Family Is Responsible for Differential Regulation of Collagenase Gene Expression by Transforming Growth Factor- in Fibroblasts and Keratinocytes (*)

(Received for publication, July 5, 1995; and in revised form, January 16, 1996)

Alain Mauviel (1) (3)(§) Kee-Yang Chung (1) (3) Akhilesh Agarwal (1) (3) Katsuto Tamai (1) (3) Jouni Uitto (1) (3) (2)

From the  (1)Departments of Dermatology and Cutaneous Biology and (2)Biochemistry and Molecular Biology, Jefferson Medical College and the (3)Section of Molecular Dermatology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor-beta (TGF-beta) plays a major role in regulating connective tissue deposition by controlling both extracellular matrix production and degradation. In this study, we show that TGF-beta transcriptionally represses both basal and tumor necrosis factor-alpha-induced collagenase (matrix metalloprotease-1) gene expression in dermal fibroblasts in culture, whereas it activates its expression in epidermal keratinocytes. We demonstrate that this differential effect of TGF-beta on collagenase gene expression is due to a cell type-specific induction of distinct oncogenes of the Jun family, which participate in the formation of AP-1 complexes with different trans-activating properties. Specifically, our data indicate that the inhibitory effect of TGF-beta in fibroblasts is likely to be mediated by jun-B, based on the following observations: (a) TGF-beta induces high levels of jun-B expression and (b) over-expression of jun-B mimics TGF-beta effect in inhibiting basal collagenase promoter activity and preventing tumor necrosis factor-alpha-induced trans-activation of the collagenase promoter. In contrast, TGF-beta induction of collagenase gene expression in keratinocytes is preceded by transient elevation of c-jun proto-oncogene expression. Over-expression of c-jun leads to trans-activation of the collagenase promoter in both cell types, suggesting that c-jun is a ubiquitous inducer of collagenase gene expression. Transfection of keratinocytes with an antisense c-jun construct together with a collagenase promoter/reporter gene construct inhibits basal and TGF-beta-induced up-regulation of the collagenase promoter activity, implying that c-jun mediates TGF-beta effect in this cell type. Collectively, our data suggest differential signaling pathways for TGF-beta in dermal fibroblasts and epidermal keratinocytes, leading to cell type-specific induction of two AP-1 components with opposite transcriptional activities.


INTRODUCTION

Matrix metalloproteases comprise a family of proteolytic enzymes involved in the degradation of the extracellular matrix of connective tissue (for reviews see (1) and (2) ). These enzymes play a critical role in a number of physiological and pathological processes involving connective tissue remodeling and/or destruction, as exemplified by embryonic development, wound repair, tumor metastasis, and rheumatoid arthritis. Breakdown of the fibrillar collagen network is initiated by interstitial collagenase (matrix metalloprotease-1), whereas the other components of the matrix are degraded primarily by stromelysins and gelatinases.

The expression of matrix metalloproteases by connective tissue cells is modulated by a variety of cytokines and growth factors(2) . In particular, interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) (^1)are potent activators of fibroblast collagenase gene expression, and their effect is mediated by c-Jun, the product of the c-jun proto-oncogene, which participates in the formation of the transcription factor AP-1(1, 2) . In contrast, transforming growth factor-beta (TGF-beta) has been shown to inhibit fibroblast collagenase gene expression through Jun-B-dependent mechanisms(3) , whereas it inhibits the expression of transin, the rat homologue of stromelysin, through Fos-mediated mechanisms(4) .

Recent in vivo observations have revealed that during cutaneous wound healing, the expression of collagenase is very low in the dermis, whereas it is markedly elevated in basal keratinocytes at the wound edges(5) . In this context, the close topographic proximity of fibroblasts and keratinocytes led us to investigate in vitro the signals that would be responsible for the differential, cell type-specific expression of collagenase during wound healing. We report that TGF-beta, a growth factor with essential wound healing promoting activities(6, 7, 8, 9) , is a potent inhibitor of collagenase gene expression in fibroblasts, whereas it strongly up-regulates collagenase expression in keratinocytes. We demonstrate that cell-specific induction of different oncogenes of the Jun family, with opposite trans-activating properties, is responsible for the differential regulation of collagenase gene expression by TGF-beta in fibroblasts and keratinocytes.


MATERIALS AND METHODS

Cell Cultures

Human dermal fibroblast cultures, established by explanting tissue specimens obtained from neonatal foreskins, were utilized in passages 3-8. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics (50 µg/ml streptomycin, 200 units/ml penicillin-G, 0.25 µg/ml Fungizone). One hour prior to the addition of growth factors, the confluent fibroblast cultures were rinsed with DMEM and placed in DMEM containing 1% fetal calf serum.

Human epidermal keratinocytes obtained by explanting foreskin specimens, were grown in serum-free, low calcium (0.15 mM), keratinocyte growth medium supplemented with epidermal growth factor, hydrocortisone, insulin, and bovine pituitary extract (KGM, Clonetics Corp., San Diego, CA), and utilized in passage 1 to avoid differentiation inherent to subculturing of these cells. One hour prior to the experiments, the confluent keratinocyte cultures were placed in fresh KGM.

Cytokines/Growth Factors

Human recombinant TGF-beta(2) was a generous gift from Dr. David R. Olsen (Celtrix Laboratories, Santa Clara, CA). Human recombinant TNF-alpha was purchased from Boehinger Mannheim.

Northern Analyses

At the end of incubation with growth factors, cell cultures were subjected to isolation of total RNA as described previously(10) . RNA was fractionated in 0.8% agarose gels containing formaldehyde and analyzed by Northern hybridization with P-labeled cDNA probes(11) . The [P]cDNA-mRNA hybrids were visualized by autoradiography, and the steady-state levels of mRNA were quantitated by scanning densitometry using a He-Ne laser scanner at 633 nm (LKB Produkter, Bromma, Sweden).

cDNAs and Plasmid Constructs

The following cDNAs were used for Northern hybridizations to detect specific mRNA transcripts: a 2.0-kilobase pair human collagenase cDNA(12) , a gift from Dr. Gregory I. Goldberg (Washington University School of Medicine, St. Louis, MO) and a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used in control hybridizations to normalize for differences in the loading and transfer of RNA(13) . For c-jun, we used a full-length human cDNA in pRSVe expression vector(14) ; and for jun-B, a full-length cDNA in pRSVe expression vector(14) , both kindly provided by Dr. Michael Karin (UCSD, La Jolla, CA).

To study the transcriptional regulation of collagenase gene expression, the following plasmid construct was used in transient transfection experiments: pCLCAT3, which contains 3.8 kilobase pairs of 5`-flanking DNA of human collagenase gene linked to the CAT reporter gene(15) , kindly provided by Dr. Steven M. Frisch (La Jolla Cancer Research Foundation, La Jolla, CA). The oncogene expression vectors described above were used in co-transfection experiments. Empty pRSVe was used as filling plasmid in order to transfect the same amount of DNA in every cell plate.

To prepare an antisense c-jun construct, a fragment spanning the region +451 to +617 of the c-jun gene was amplified by polymerase chain reaction (PCR). The PCR amplimers were cloned into a PCRII plasmid vector (InVitrogen Corp., San Diego, CA), and the clones containing the insert were sequenced to ensure the fidelity of the PCR amplification. The PCR products were then inserted in an antisense orientation as XhoI-HindIII fragments into the pRSVe expression vector in order to generate the construct pRSV-ASc-jun.

Transient Transfections of Cultured Cells

Human neonatal foreskin fibroblasts in late logarithmic growth phase were transfected with 1-20 µg of plasmid constructs co-transfected with a RSV-promoter/beta-galactosidase construct to allow determination of the transfection efficiency(11) . The transfections were performed with the calcium-phosphate/DNA co-precipitation method (16) followed by a 1.5-min glycerol (15%) shock. Following the glycerol shock, the cells were placed in medium supplemented with 1% fetal calf serum prior to the addition of growth factors. Basal keratinocytes grown in KGM were transfected with a liposome-based method (DOTAP, Boehringer Mannheim), according to the manufacturer's protocol. Five hours after transfections, the medium was replaced with fresh KGM, and growth factors were added 2 h later for a 40-h incubation. At the end of incubation, the cells were harvested and lysed by thee cycles of freeze-thawing in 100 µl of 0.25 M Tris-HCl, pH 7.8. Aliquots corresponding to identical beta-galactosidase activity were used for each CAT assay with [^14C]chloramphenicol as substrate(17) .

Stable Transfections

To generate stably transfected cultures of NIH3T3 fibroblasts, pRSV-ASc-jun was co-transfected with pRc/CMV (InVitrogen), which expresses the neomycin resistance gene, in a 10:1 ratio. Four days after transfections, G418 (800 µg/ml, Life Technologies, Inc.) was added to the culture medium for selection of transfected cells. Fifteen days later, stably transfected cultures of pRSV-ASc-jun NIH3T3 were established by pooling the G418-resistant clones. Control cultures were transfected with pRc/CMV and empty pRSVe to generate pRSVeNIH3T3 cultures.


RESULTS

Cell Type-specific Effect of TGF-beta on Collagenase Gene Expression

In the first set of experiments, human adult skin fibroblasts were incubated for 24 h without or with TGF-beta or TNF-alpha, both at a concentration of 10 ng/ml. At the end of incubation, RNA was extracted and analyzed for collagenase gene expression by Northern hybridization. Visual observation of the autoradiograms indicated that TGF-beta markedly down-regulated both basal and TNF-alpha-induced elevation of collagenase mRNA steady-state levels in parallel fibroblast cultures (Fig. 1A). Quantitation of the autoradiograms by scanning densitometry and correction against GAPDH mRNA levels in the corresponding RNA preparations indicated that the expression of collagenase in TGF-beta-treated cultures was reduced by 80% in comparison with control fibroblast cultures. TNF-alpha enhanced collagenase mRNA levels by 6-fold, but in cultures treated concomitantly with TNF-alpha and TGF-beta, the levels were reduced by 75% in comparison with cultures treated with TNF-alpha alone (Fig. 1B).


Figure 1: Effect of TGF-beta on collagenase gene expression in dermal fibroblasts. Confluent fibroblast cultures were incubated in medium containing 1% fetal calf serum without(-) or with (+) TNF-alpha (10 ng/ml), in the absence(-) or the presence (+) of TGF-beta (10 ng/ml). After 24 h, total RNA was extracted and analyzed by Northern hybridization with a collagenase-specific cDNA; a GAPDH cDNA was used as a control. A, autoradiograms. B, densitometric analysis after correction for GAPDH mRNA levels.



In the second set of experiments, confluent keratinocyte cultures were incubated for 24 h without or with TGF-beta and/or TNF-alpha at concentrations of either 1 or 10 ng/ml. Contrasting with its inhibitory effect observed in dermal fibroblasts, TGF-beta was found to be a potent enhancer of keratinocyte collagenase gene expression (Fig. 2, second and third lanes). On the other hand, TNF-alpha, which is a potent enhancer of collagenase gene expression in fibroblasts (see Fig. 1), had little effect on the basal expression of collagenase in keratinocytes (Fig. 2, fifth and sixth lanes) and a minimal effect on the induction exerted by TGF-beta (Fig. 2, fourth lane). Quantitation of the autoradiograms after normalization of collagenase mRNA levels against those of GAPDH showed a 4.5-5-fold elevation of collagenase mRNA steady-state levels upon TGF-beta stimulation, whereas TNF-alpha did not stimulate collagenase expression (Fig. 2B).


Figure 2: Effect of TGF-beta on collagenase gene expression in epidermal keratinocytes. Confluent keratinocyte cultures in serum-free KGM were incubated for 24 h in the absence or the presence of TGF-beta without or with TNF-alpha in the concentrations indicated. After 24 h, total RNA was extracted and analyzed by Northern hybridization with a collagenase-specific cDNA; a GAPDH cDNA was used as a control. A, autoradiograms. B, densitometric analysis after correction for GAPDH mRNA levels.



Additional experiments with various concentrations (0.1, 1, and 10 ng/ml) of TGF-beta indicated a stimulatory effect with as little as 0.1 ng/ml (2.8-fold); whereas maximal elevation of collagenase mRNA levels (6-fold) was observed with 1 ng/ml of TGF-beta, no further enhancement was noted with 10 ng/ml of TGF-beta (not shown).

Transient cell transfections with a collagenase promoter/CAT reporter gene construct were performed to examine whether TGF-beta regulates collagenase mRNA steady-state levels through modulation of transcription at the promoter level. Human neonatal fibroblasts were transfected with the collagenase promoter/CAT construct pCLCAT3 and treated with TGF-beta or TNF-alpha both at 10 ng/ml concentration. Assay of CAT activity after 40 h of incubation indicated that TGF-beta reduced the promoter activity by 40-60%, as compared with that of control cultures (Fig. 3). Also TGF-beta counteracted TNF-alpha-induced elevation of the collagenase promoter activity.


Figure 3: Effect of TGF-beta on collagenase promoter activity in dermal fibroblasts. Fibroblasts in late logarithmic growth phase were transfected with 10 µg/plate of collagenase promoter/CAT (pCLCAT3) plasmid construct as described under ``Materials and Methods.'' Following the glycerol shock, the cells were placed in medium supplemented with 1% fetal calf serum. Three hours later, TNF-alpha and/or TGF-beta, both at a concentration of 10 ng/ml, were added (+) and incubations were continued for 40 h. CAT activity, representing collagenase promoter activity, was determined. The results, presented as relative promoter activity, are the means ± S.D. of four independent experiments, each point run with duplicate samples and expressed as fold induction over the controls, which are set as 1.0.



In another set of experiments, confluent keratinocyte cultures were transfected with the same collagenase promoter/CAT reporter gene construct and treated with various doses of TGF-beta (0.1, 1, and 10 ng/ml) for 40 h. Assay of CAT activity revealed a dose-dependent elevation of the promoter activity (Fig. 4) with a maximal stimulation (4-fold) observed with 10 ng/ml of TGF-beta.


Figure 4: Effect of TGF-beta on collagenase promoter activity in epidermal keratinocytes. Confluent keratinocyte cultures in serum-free KGM were transfected with pCLCAT3 using a liposome-based method (DOTAP, Boehinger Mannheim) according to the manufacturer's protocol. Five hours after transfections, the medium was replaced with fresh KGM and TGF-beta in various concentrations was added 2 h later for a 40-h incubation. CAT activity, representing collagenase promoter activity, was determined. The results, presented as relative promoter activity, are the means ± S.D. of three independent experiments run with duplicate samples and expressed as fold induction over the controls, which are set as 1.0.



Thus, the enhancement of collagenase gene expression in keratinocytes by TGF-beta and its inhibition in fibroblasts, as detected at the mRNA level, was mediated, at least in part, by cell was type-specific modulation of the promoter activity.

Cell Type-specific Induction of jun-B and c-jun Proto-oncogene Expression by TGF-beta

It has been previously reported that the expression of c-jun is induced by cytokines such as TNF-alpha and interleukin-1 (18, 19) and represents a fundamental intermediate step in cytokine induction of other cellular genes, including those encoding collagenase or stromelysin (reviewed in (1) and (2) ).

As shown in Fig. 5, TNF-alpha rapidly (within 1 h of incubation) enhanced the expression of c-jun in dermal fibroblasts (lane 4), and the induction persisted even after 6 h of incubation (lane 5). Also, the steady-state levels of jun-B mRNAs were elevated but to a lesser extent than those for c-jun (lane 4). TGF-beta alone did not affect either the basal expression of c-jun (lane 7) or the induction of c-jun by TNF-alpha (lanes 9 and 10 versus lanes 4 and 5). In contrast, TGF-beta, either alone or in combination with TNF-alpha, strongly elevated the expression of jun-B (lanes 7 and 9), which persisted for at least up to 6 h following the initiation of stimulation (lanes 8 and 10). It appears, therefore, that in fibroblasts, TGF-beta counteracts TNF-alpha-induced collagenase gene expression, but this effect is not due to repression of c-jun transcription. At the same time, TNF-alpha did not alter the expression of jun-B induced by TGF-beta. Accordingly, TGF-beta stimulation led to a dramatic reduction of the c-jun/jun-B mRNA ratio in fibroblasts, from 1.7 to 0.1 and 0.2 after 1 and 6 h of stimulation, respectively (Fig. 6). In contrast, TNF-alpha elevated the c-jun/jun-B mRNA ratio due to a substantially higher stimulation of c-jun versus jun-B. When TGF-beta was added simultaneously with TNF-alpha, the c-jun/jun-B mRNA ratio remained at levels that were 60-70% lower than observed with TNF-alpha alone (Fig. 6). Therefore, there is a strong correlation between the c-jun/jun-B mRNA ratio and the level of collagenase expression, suggesting that the modification of the ratio of c-jun/jun-B mRNA is likely to reflect a reduction of the relative amounts of c-Jun versus those of Jun-B when TGF-beta is present, leading to reduced collagenase gene transcription.


Figure 5: Effect of TNF-alpha and TGF-beta on c-jun and jun-B gene expression in dermal fibroblasts. Confluent fibroblast cultures were incubated in medium containing 1% fetal calf serum without (CTL) or with TNF-alpha (10 ng/ml), in the absence(-) or the presence (+) of TGF-beta (10 ng/ml). Total RNA was extracted after 0, 1, or 6 h of incubation and analyzed by Northern hybridizations with P-labeled cDNA probes specific for c-jun and jun-B mRNAs. A GAPDH cDNA was used as a control.




Figure 6: Effect of TGF-beta on the ratio of c-jun/jun-B mRNA in dermal fibroblasts. Relative c-jun and jun-B mRNA levels were determined in each RNA preparation by densitometric analysis of the autoradiograms shown in Fig. 5. Raw values were corrected for GAPDH mRNA levels in the same RNA preparations and for the specific activity of the c-jun and jun-B probes. The ratio of c-jun and jun-B mRNA levels was calculated for the various time points of cytokine treatment. Open squares, control; solid squares, TGF-beta; open triangles, TNF-alpha; solid triangles, TNF-alpha + TGF-beta.



Subsequently, we tested the pattern of expression of oncogenes of the Jun family in the presence of TGF-beta in epidermal keratinocytes. Contrasting with its lack of effect in fibroblasts, TGF-beta induced high levels of c-jun expression in keratinocytes, with a maximum enhancement at 1 h following growth factor stimulation (Fig. 7). The high levels of c-jun mRNAs persisted at least 6 h post-stimulation and preceded the induction of collagenase expression. In contrast, the basal expression of jun-B was very low and, although expression was enhanced after 1 h of incubation with TGF-beta, it remained well below the level of expression of c-jun. In fact, using P-labeled cDNA probes with similar specific activities (2 times 10^7 cpm/µg), a 48-h exposure of the autoradiogram was necessary to detect the low jun-B expression, whereas a 14-h exposure was sufficient to easily detect c-jun expression (Fig. 7). Therefore, in keratinocytes, after TGF-beta stimulation, the ratio c-jun/jun-B is largely in favor of c-jun, 25-fold more c-jun mRNA than jun-B at 1 h after addition of TGF-beta, as estimated by scanning densitometry of the autoradiograms, correction for GAPDH mRNA levels in the RNA preparations, and differences in the exposure times of the autoradiograms. These data contrast the reverse situation observed in dermal fibroblasts in which jun-B expression is boosted by TGF-beta treatment (see above, Fig. 5and Fig. 6). It should be noted that the extent of stimulation of collagenase gene expression as detected at the mRNA level (15-fold after 6 h) was more pronounced than the induction of promoter activity observed in transient cell transfection experiments (see Fig. 4). It is conceivable that TGF-beta, in addition to activating the collagenase promoter, may also increase collagenase gene expression by post-transcriptional mechanisms such as stabilization of the corresponding mRNA, as described previously for phorbol esters or epidermal growth factor(20, 21) .


Figure 7: Effect of TGF-beta on c-jun and jun-B gene expression in epidermal keratinocytes. Confluent keratinocyte cultures were incubated in serum-free KGM with TGF-beta (10 ng/ml). Total RNA was extracted at various time points following the addition of TGF-beta and analyzed by Northern hybridizations with cDNAs specific for collagenase and c-jun and jun-B mRNAs. A GAPDH cDNA was used as a control. The specific activities of the probes, 2 times 10^7 cpm/µg, differed by less than 15%. Exposure times of the different hybridizations were 14 h for c-jun, 48 h for jun-B, and 24 h for collagenase.



c-jun Trans-activates the Collagenase Promoter in Both Fibroblasts and Keratinocytes

In view of the data described above, it appears that in both cell types studied, enhanced collagenase gene expression correlates with a preceding elevation of c-jun mRNA levels due to TNF-alpha stimulation in fibroblasts and to TGF-beta stimulation in keratinocytes, respectively. We therefore tested whether over-expression of c-jun could trans-activate the collagenase promoter in both cell types. For this purpose, pCLCAT3 was co-transfected with either an RSV/c-jun expression vector or an empty RSVe vector as a control. Over-expression of c-jun in both fibroblasts and keratinocytes led to trans-activation of the collagenase promoter, as reflected by measurement of CAT activity in the different cell extracts. The extent of stimulation of the collagenase promoter was similar in both cell types: 5.3 ± 0.7-fold (n = 8) in fibroblasts versus 4.9 ± 0.9-fold (n = 6) in keratinocytes. In contrast, over-expression of jun-B resulted in reduced collagenase promoter activity in both cell types (not shown), attesting to the specificity of the expression vectors, as expected from previous observations by us and others(3, 14) .

Characterization of the Antisense Activity of pRSV-ASc-jun

Control (pRSVeNIH3T3) and pRSV-ASc-junNIH3T3 fibroblast cultures (see ``Materials and Methods'') were grown to confluency. Three hours prior to the addition of growth factors, the confluent fibroblast cultures were rinsed with DMEM and placed in DMEM containing 1% fetal calf serum. The cultures were then treated for 6 h with either TNF-alpha or TGF-beta, both at a concentration of 10 ng/ml, prior to Northern analysis. As shown in Fig. 8, significant expression of both c-jun and jun-B was noted in unstimulated pRSVeNIH3T3 cultures (lane 1). TNF-alpha strongly elevated c-jun expression, and jun-B to a lesser extent (lane 2). TGF-beta slightly elevated jun-B expression but had no effect on c-jun mRNA levels (lane 3). pRSV-ASc-junNIH3T3 cultures showed very little basal expression of c-jun (lanes 4), about 7% of that observed in control cultures, as measured by scanning densitometry and after correction for GAPDH mRNA levels in the same RNA preparations, whereas the basal levels of jun-B mRNA were similar to those of control cultures. TNF-alpha did not elevate c-jun mRNAs in pRSV-ASc-jun-transfected cultures, but the induction of jun-B by both TNF-alpha and TGF-beta was similar to that observed in control cultures (Fig. 8, fifth and sixth lanes versus first and second lanes), indicating that the cells are still responsive to growth factors. Taken together, these results demonstrate that pRSV-ASc-jun prevents both basal and TNF-alpha-induced c-jun expression, whereas the construct has no effect on the expression and regulation of jun-B expression. Interestingly, no expression of collagenase was detected in pRSV-ASc-junNIH3T3 cultures, even after TNF-alpha stimulation, indicating that c-jun is fundamental for both basal and cytokine-induced collagenase gene expression. Also, these data demonstrate that the c-jun antisense construct can block c-Jun-mediated transcription.


Figure 8: Characterization of pRSV-ASc-jun in stable transfection experiments. In order to verify the activity of the pRSV-ASc-jun antisense construct, pRSVeNIH3T3 and pRSV-ASc-junNIH3T3 fibroblast cultures were grown to confluency. Three hours prior to the addition of growth factors, the confluent fibroblast cultures were rinsed with DMEM and placed in DMEM containing 1% fetal calf serum. The cultures were then treated for 6 h with either TNF-alpha or TGF-beta, each at a concentration of 10 ng/ml. Total RNA was extracted and analyzed by Northern hybridization with P-labeled cDNA probes for collagenase, c-jun and jun-B. A GAPDH cDNA was used as a control.



Antisense c-jun Prevents TGF-beta-induced Elevation of Collagenase Promoter Activity in Epidermal Keratinocytes

We have shown that TGF-beta stimulates c-jun expression prior to inducing collagenase gene expression in keratinocytes and that over-expression of c-jun by the mean of transfected expression vectors leads to trans-activation of the collagenase promoter in keratinocytes (see above). We therefore wished to examine whether the stimulatory effect of TGF-beta was directly mediated by c-jun. For this purpose, pRSV-ASc-jun was co-transfected with pCLCAT3 into basal keratinocytes, prior to stimulation with TGF-beta. As shown in Fig. 9, the basal activity of the collagenase promoter was markedly reduced by the antisense c-jun (by about 70%), which implies a role for c-jun in the basal expression of collagenase in keratinocytes. Furthermore, antisense c-jun prevented stimulation of collagenase promoter activity by TGF-beta (Fig. 9), suggesting that c-jun induction is an essential step for collagenase gene activation by TGF-beta in keratinocytes.


Figure 9: Effect of antisense c-jun on TGF-beta-mediated up-regulation of collagenase promoter activity in keratinocytes. Confluent keratinocyte cultures were transfected with 3 µg/plate of pCLCAT3, together with 17 µg/plate of either pRSVe or pRSV-ASc-jun. After 18 h, medium was replaced with fresh KGM. Six hours later, TGF-beta (10 ng/ml) was added to the cultures (+). Incubations were continued for 40 h, and CAT activity, representing the promoter activity, was determined. The results are the means ± S.D. of three independent experiments.




DISCUSSION

Several studies have shown differences in the pattern of expression and response to extracellular stimuli between c-jun and jun-B in a variety of experimental systems(3, 22, 23, 24) . Furthermore, considerable differences in their trans-activation and transforming activities have been reported(3, 14, 25, 26) . For example, whereas c-Jun is an efficient activator of the c-jun and collagenase promoters that contain a single TRE, Jun-B is not. In addition, Jun-B counteracts activation of these promoters by c-Jun. However, like c-Jun, Jun-B is a potent activator of constructs containing multimeric TREs. These differences in the biological actions of the two Jun proteins are due to intrinsic differences in their activation and DNA-binding domains (14, 27) , allowing fine tuning of the regulation of TRE-driven genes.

The regulatory role of the different Jun proteins is further emphasized by the fact that the corresponding genes are not coordinately expressed in different tissues, as shown in adult mice and during embryogenesis (24, 28, 29) , suggesting a tissue-specific transcriptional regulation of TRE-driven target genes. In this context, it has been recently shown that jun-B and c-jun are selectively up-regulated and functionally implicated in the development of fibrosarcoma(30) . It is therefore conceivable that in different inducible systems, increased specificity and precise regulation of TRE-driven transcriptional activation is achieved by interactions between positive and negative transcription factors that belong to the same gene family.

In this study, we have provided the following evidence for a mediation of the inhibitory effect of TGF-beta on fibroblast collagenase gene expression by Jun-B: (a) TGF-beta induces high levels of jun-B expression; (b) jun-B expression vectors mimic TGF-beta action in our experimental system by inhibiting basal collagenase promoter activity and by exerting an antagonistic effect on TNF-alpha-induced collagenase gene expression. On the other hand, we have demonstrated that TGF-beta stimulates collagenase gene expression in keratinocytes and that this effect is mediated by c-jun, as follows: (a) TGF-beta induces high levels of c-jun expression; (b) c-jun expression vectors trans-activate the collagenase promoter; (c) antisense c-jun expression vectors prevent TGF-beta activation of collagenase gene expression. A schematic diagram depicting the differential effects of TGF-beta on collagenase gene expression in fibroblasts and keratinocytes is shown in Fig. 10. These results are the first evidence of differential induction of two oncogenes with opposite trans-activation properties upon stimulation by a single growth factor, TGF-beta, in two different cell types within the same tissue, the skin.


Figure 10: Schematic representation of the putative mechanisms for differential regulation of collagenase gene expression by TGF-beta in fibroblasts and keratinocytes. Left, TGF-beta, through interactions with specific receptors on the keratinocyte surface, induces high levels of expression of c-jun, which, after dimerization, is responsible for the trans-activation of the collagenase promoter. Right, TGF-beta induces high levels of expression of jun-B, and its product participates in the formation of AP-1 complexes with inhibitory activity on collagenase gene expression. Transduction mechanisms (1; to be identified) induce transcription of c-jun in keratinocytes and of jun-B in fibroblasts (2). Jun products are translated in the cytoplasm (3) and translocate into the nucleus (4) to form AP-1 complexes that modulate collagenase gene transcription (5). TGF-beta-induced c-jun expression in keratinocytes leads to increased collagenase gene transcription (+) and increased collagenase production (&cjs3832;&cjs3832;). In fibroblasts, increased jun-B expression represses collagenase transcription(-) with subsequent decrease in collagenase production (&cjs3706;&cjs3706;).



TGF-beta has been shown to reduce collagenase gene expression and activity in cultured fibroblasts. This inhibition results from two distinct mechanisms: (a) TGF-beta reduces the expression of the collagenase gene and (b) the expression of tissue inhibitor of metalloproteases is elevated by TGF-beta(31) . Our data indicate that the concept of TGF-beta as a potent inhibitor of matrix remodeling is cell type-specific because this growth factor is a potent activator of collagenase gene expression in epidermal keratinocytes in culture. In that respect, TGF-beta has been shown previously to up-regulate both 92- and 72-kDa gelatinase activity and gene expression in both fibroblasts and keratinocytes(32, 33) .

Recently, it has been demonstrated using in situ hybridization in ulcerative skin lesions such as pyogenic granulomas that collagenase is expressed near the advancing edge of the ulceration, within the disrupted epidermis adjacent to an ulcer(5) . By contrast, no hybridization signal was detected within the dermis or normal, intact epidermis. Therefore, basal keratinocytes seem to be the primary source of collagenase during wound healing, suggesting that keratinocytes play an essential role in tissue remodeling. It has been suggested that the signals that activate collagenase in keratinocytes are provided by the dermal extracellular matrix. In agreement with this hypothesis is the fact that keratinocytes grown on type I collagen exhibit enhanced collagenase production(34) . By contrast, activation of fibroblast collagenase expression may be mediated by soluble factors such as interleukin-1 or TNF-alpha, rather than by the extracellular matrix. Skin injury is accompanied by release of interleukin-1, which in turn may activate fibroblasts but not keratinocytes to produce collagenase(34) . Our data provide an alternative model for the cell-specific activation of collagenase gene expression during wound healing in which TGF-beta, which is present in abundant amounts in the healing wound bed, could simultaneously turn off the expression of collagenase in fibroblasts while activating that of keratinocytes directly in contact with the dermis. We hypothesize that collagenase-secreting keratinocytes, possibly in response to TGF-beta, may be able to migrate to close the wound. This hypothesis is supported indirectly by a previous study indicating that TGF-beta stimulates the outgrowth of epidermal cells from skin explant cultures(35) .

In conclusion, this study has provided the first evidence for cell type-specific, differential induction of two transcription factors of the same family with antagonistic trans-activation properties, leading to opposite regulation of collagenase gene expression in fibroblasts and keratinocytes by TGF-beta.


FOOTNOTES

*
This work was supported in part by the United States Public Health Service, National Institutes of Health Grants RO1-AR41439 and T32-AR07561 (to J. U.) and by a Dermatology Foundation Research Career Development Award (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Thomas Jefferson University, Dept. of Dermatology and Cutaneous Biology, 233 South 10th St., Rm. 430, Philadelphia, PA 19107. Tel.: 215-955-5775; Fax: 215-923-9354.

(^1)
The abbreviations used are: TNF-alpha, tumor necrosis factor-alpha; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KGM, keratinocyte growth medium; TGF-beta, transforming growth factor-beta; CAT, chloramphenical acetyltransferase; PCR, polymerase chain reaction; RSV, Rous sarcoma virus; TRE, TPA response element.


ACKNOWLEDGEMENTS

We express our gratitude to Drs. Michael Karin, David R. Olsen, Gregory I. Goldberg, and Steven M. Frisch, who provided constructs and reagents essential for completion of this study, to Drs. John Moore, Jr., and James Fox IV, who kindly provided tissue for cell cultures, and to Lin Lin for expert technical assistance.


REFERENCES

  1. Woessner, J. F., Jr. (1991) FASEB J. 5, 2145-2154 [Abstract/Free Full Text]
  2. Mauviel, A. (1993) J. Cell. Biochem. 53, 288-295 [Medline] [Order article via Infotrieve]
  3. Mauviel, A., Chen, Y. Q., Dong, W., Evans, C. H., and Uitto, J. (1993) Curr. Biol. 3, 822-831
  4. Kerr, L. D., Miller, D. B., and Matrisian, L. M. (1990) Cell 61, 267-278 [Medline] [Order article via Infotrieve]
  5. Saarialho-Kere, U., Chang, E. S., Welgus, H. G., and Parks, W. C. (1992) J. Clin. Invest. 90, 1952-1957 [Medline] [Order article via Infotrieve]
  6. Barnard, J. A., Lyons, R. M., and Moses, H. L. (1990) Biochim. Biophys. Acta 1032, 79-87 [CrossRef][Medline] [Order article via Infotrieve]
  7. Massagué, J. (1990) Annu. Rev. Cell Biol. 6, 597-641 [CrossRef]
  8. Roberts, A. B., and Sporn, M. B. (1990) in Peptide Growth Factors and Their Receptors (Sporn, M. B., and Roberts, A. B., eds) pp. 419-472, Springer-Verlag New York Inc., New York
  9. Wahl, S, M. (1992) J. Clin. Immun. 12, 61-74 [Medline] [Order article via Infotrieve]
  10. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  11. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Second Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  12. Goldberg, G. I., Wilhelm, S. M., Kronberger, A., Bauer, E. A., Grant, G. A., and Eisen, A. Z. (1986) J. Biol. Chem. 261, 6600-6605 [Abstract/Free Full Text]
  13. Fort, P., Marty, L., Piechaczyk, M., El Sabrouty, S., Danz, C., Jeanteur, P., and Blanchard, J.-M. (1985) Nucleic Acids Res. 13, 1431-1442 [Abstract]
  14. Chiu, R., Angel, P., and Karin, M. (1989) Cell 59, 979-986 [Medline] [Order article via Infotrieve]
  15. Frisch, S. M., Reich, R., Collier, I. E., Genrich, L. T., Martin, G., and Goldberg, G. I. (1990) Oncogene 5, 75-83 [Medline] [Order article via Infotrieve]
  16. Graham, F., and Van der Eb, A. (1973) Virology 52, 456-457 [Medline] [Order article via Infotrieve]
  17. Gorman, C. M., Moffat, L. F., Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051 [Medline] [Order article via Infotrieve]
  18. Brenner, D. A., O'Hara, M., Angel, P., Chojkier, M., and Karin, M. (1989) Nature 337, 661-663 [CrossRef][Medline] [Order article via Infotrieve]
  19. Conca, W., Kaplan, P. B., and Krane, S. M. (1989) J. Clin. Invest. 83, 1753-1757 [Medline] [Order article via Infotrieve]
  20. Brinckerhoff, C. E., Plucinska, I. M., Sheldon, L. A., and O'Connor, G. T. (1986) Biochemistry 25, 6378-6384 [Medline] [Order article via Infotrieve]
  21. Delany, A. M., and Brinckerhoff, C. E. (1992) J. Cell. Biochem. 50, 400-410 [Medline] [Order article via Infotrieve]
  22. Bartel, D., Sheng, M., Lau, L., and Greenberg, M. (1989) Genes & Dev. 3, 304-313
  23. Pertovaara, L., Sistonen, L., Bos, T., Vogt, P., Keski-Oja, J., and Alitalo, K. (1989) Mol. Cell. Biol. 9, 1255-1262 [Medline] [Order article via Infotrieve]
  24. Wilkinson, D. G., Bhatt, S., Ryseck, R. P., and Bravo, R. (1989) Development 106, 465-471 [Abstract]
  25. Schütte, J., Viallet, J., Nau, M., Segal, S., Fedorko, J., and Minna, J. (1989) Cell 59, 987-997 [Medline] [Order article via Infotrieve]
  26. Ryseck, R. F., and Bravo, R. (1991) Oncogene 6, 533-542 [Medline] [Order article via Infotrieve]
  27. Deng, T., and Karin, M. (1993) Genes & Dev. 7, 479-490
  28. Hirai, S. I., Ryseck, R. P., Mechta, F., Bravo, R., and Yaniv, M. (1989) EMBO J. 8, 1433-1438 [Abstract]
  29. Mellström, B., Achaval, M., Montero, D., Navanjo, J. R., and Sassone-Corsi, P. (1991) Oncogene 6, 1959-1964 [Medline] [Order article via Infotrieve]
  30. Bossy-Wetzel, E., Bravo, R., and Hanahan, D. (1992) Genes & Dev. 6, 2340-2351
  31. Edwards, D. R., Murphy, G., Reynolds, J. J., Whitham, S. E., Docherty, A. J. P., Angel, P., and Heath, J. K. (1987) EMBO J. 6, 1899-1904 [Abstract]
  32. Overall, C. M., Wrana, J. L., and Sodek, J. (1991) J. Biol. Chem. 266, 14064-14071 [Abstract/Free Full Text]
  33. Salo, T., Lyons, J. G., Rahemtulla, F., Birkedal-Hansen, H., and Larjava, H. (1991) J. Biol. Chem. 266, 11436-11441 [Abstract/Free Full Text]
  34. Petersen, M. J., Woodley, D. T., Stricklin, G. P., and O'Keefe, E. J. (1990) J. Invest. Dermatol. 97, 341-346
  35. Hebda, P. (1988) J. Invest. Dermatol. 91, 440-445 [Abstract]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.