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Expression of human collagenase-3 (MMP-13) by fetal skin fibroblasts is induced by transforming growth factor ß via p38 mitogen-activated protein kinase¹

LAURA RAVANTI^*,,, MERVI TORISEVA^*, RISTO PENTTINEN, TIMOTHY CROMBLEHOLME, MARCO FOSCHI^**, JIAHUAI HAN and VELI-MATTI KÄHÄRI^*,,2

^* Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FIN-20520 Turku, Finland;
Department of Medical Biochemistry, University of Turku, FIN-20520 Turku, Finland;
Department of Dermatology, Turku University Central Hospital, FIN-20520 Turku, Finland;
The Children’s Institute for Surgical Science, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA;
^** Department of Internal Medicine, University of Florence, Florence 50134, Italy; and
Department of Immunology, Scripps Research Institute, La Jolla, California 92121, USA

²Correspondence: Turku Centre for Biotechnology, University of Turku, Tykistökatu 6B, FIN-20520 Turku, Finland. E-mail: veli-matti.kahari{at}btk.utu.fi

SPECIFIC AIMS

In the present study we have examined the possible role of collagenase-3 (matrix metalloproteinase-13; MMP-13), a collagenolytic MMP with a wide substrate specificity, in human fetal skin wound repair characterized by minimal scar formation. The expression of MMP-13 was examined in wounds in human fetal skin grafted on SCID mice, and the regulation of human MMP-13 expression was examined in fetal skin fibroblasts in culture.

PRINCIPAL FINDINGS

1. Human MMP-13 is expressed by fibroblasts in fetal skin wounds
To elucidate the role and regulation of human MMP-13 in fetal wound repair, which is characterized by minimal scar formation, we first examined the expression of MMP-13 in a well-characterized model of normally healing incisional wound of human fetal skin (16 to 20 wk of gestational age) grafted on SCID mice. MMP-13-positive fibroblasts were detected by immunostaining within the dermal layer in 4-day-old wounds (Fig. 1A , B ). Numerous MMP-13-positive dermal fibroblasts were also detected in the margin of the fetal skin graft, whereas dermal fibroblasts in adjacent murine skin were negative for MMP-13, confirming the specificity of the antibody for human MMP-13 (Fig. 1C ). These observations demonstrate a remarkable difference in MMP-13 expression between normally healing fetal and adult cutaneous wounds, suggesting that the regulation of MMP-13 expression in fetal and adult human skin fibroblasts is fundamentally different.

View larger version (55K): [in this window] [in a new window]   Figure 1. Expression of human collagenase-3 (MMP-13) by fibroblasts in fetal skin wounds. Human fetal skin of gestational age 16 to 20 wk was grafted onto SCID mice and wounded, as described in Materials and Methods. Wound samples obtained at 4 days were immunostained with anti-MMP-13 antibody, which does not cross-react with murine MMP-13. A) the margins of newly epithelialized wound are marked with arrows; B) MMP-13-positive dermal fibroblasts are indicated by arrowheads; C) MMP-13-positive fibroblasts are detected in human fetal dermal layer (HF), but not in murine dermal layer (M). Bars: A, 100 µM; B, C, 20 µM.

2. Expression of MMP-13 in fetal skin fibroblasts is induced by transforming growth factor ß (TGF-ß)
To examine the regulation of MMP-13 expression in fetal skin fibroblasts, we incubated fibroblasts from human fetal skin at gestational age of 17 wk and from neonatal skin without or with TGF-ß1 (5 ng/ml) for 48 h, and determined the levels of MMP-13 in conditioned media by Western blot analysis. Fetal skin fibroblasts produced proMMP-13 when treated with TGF-ß1, whereas neonatal skin fibroblasts in monolayer culture did not produce detectable amounts of proMMP-13. In fetal skin fibroblasts, collagenase-1 (MMP-1) production was not markedly altered by TGF-ß1, whereas in neonatal skin fibroblasts proMMP-1 production was down-regulated by TGF-ß1, as in human adult skin fibroblasts. TIMP-1 production was enhanced by TGF-ß1 in both fetal and neonatal skin fibroblasts, corroborating their response to TGF-ß1. Treatment with TGF-ß1 and TGF-ß3 enhanced the abundance of MMP-13 mRNAs in fetal skin fibroblasts, but not in neonatal skin fibroblasts.

3. Induction of MMP-13 expression in fetal skin fibroblasts by TGF-ß1 is mediated by p38 MAPK
Treatment of human gingival fibroblasts with TGF-ß1 rapidly and transiently activates two mitogen-activated protein kinases—extracellular signal-regulated kinase (ERK) 1,2 and p38—and p38 MAPK activity is required for induction of MMP-13 expression by TGF-ß1. To study the role of MAPK signaling pathways in the regulation of human fetal skin fibroblast MMP-13 expression, we first determined the activation of these MAPKs by Western blot analysis of cellular proteins at various time points after exposure to TGF-ß1 using antibodies against the active, phosphorylated forms of these MAPKs. The levels of activated p38 were increased 3.8-fold at 30 min of incubation with TGF-ß1, and a potent induction (6.1-fold) was still noted at 6 h of incubation. Treatment with TGF-ß1 did not result in activation of ERK1,2 in fetal skin fibroblasts. In contrast, ERK1,2 was rapidly activated (2.4-fold) at 15 min of incubation with TGF-ß1 in neonatal skin fibroblasts. In addition, p38 was activated maximally (2.2-fold) at 1 h after addition of TGF-ß1.

To study the specific roles of MAPKs in mediating the induction of MMP-13 expression by TGF-ß1 in fetal skin fibroblasts, we first used selective chemical inhibitors for ERK1,2 and p38 MAPK. Blocking the ERK1,2 pathway (RafMEK1,2ERK1,2) by PD98059 (30 µM), a specific inhibitor of MEK1,2 added to fibroblasts 1 h prior to TGF-ß1, had no effect on the induction of proMMP-13 production by TGF-ß1 (Fig. 2A ). In contrast, addition of selective p38 inhibitor SB203580 (10 µM) to fibroblasts 1 h before TGF-ß1 inhibited completely the production of proMMP-13 (Fig. 2A ). Treatment of fetal skin fibroblasts with TGF-ß1 without or with PD98059 or SB203580 had no marked effect on proMMP-1 production. TIMP-1 production was slightly (1.5-fold) enhanced by TGF-ß1, but this stimulation was not markedly altered by PD98059 or SB203580.

View larger version (27K): [in this window] [in a new window]   Figure 2. Induction of collagenase-3 (MMP-13) production by fetal skin fibroblasts by TGF-ß is dependent on the activity of p38 MAPK. A) Human fetal skin fibroblasts (wk 17) were incubated with TGF-ß1 (5 ng/ml) for 48 h in serum-free DMEM. PD98059 (30 µM), a specific inhibitor of ERK1,2 kinases MEK1,2, or SB203580 (10 µM), a selective p38 inhibitor, were added to cultures (+) 1 h prior to TGF-ß1. The levels of proMMP-13, proMMP-1, and TIMP-1 in conditioned media of fetal skin fibroblasts were determined by Western blot analysis. B) Human fetal skin fibroblasts (wk 17) were infected at MOI 500 with control adenovirus (RAd66) and with adenoviruses for dominant negative Rac1 (RAdN17rac1) and dominant negative p38 (RAdp38AF), and incubated for 5 h in DMEM with 1% FCS. Thereafter, medium was replaced with serum-free DMEM without (-) or with (+) TGF-ß1 (5 ng/ml) and incubations were continued for 24 h. The levels of proMMP-13 and TIMP-1 in the conditioned media were determined by Western blot analysis.

To further elucidate the role of MAPK signaling pathways in mediating the induction of MMP-13 gene expression by TGF-ß1, we used replication-deficient adenoviruses to specifically inhibit and activate endogenous MAPK cascades. Determination of the transduction efficiency of human fetal skin fibroblasts using adenovirus RAdlacZ showed that the ß-galactosidase gene was delivered to all cells at MOI 500, which was then used in experiments. We infected the cells with adenoviruses coding for dominant negative forms of small GTPase Rac1 (RAdN17rac1), involved in activation of JNK and p38, and p38 (RAdp38AF). Treatment of fetal skin fibroblasts with TGF-ß1 resulted in induction in proMMP-13 production in cells infected with the empty control virus RAd66 (Fig. 2B ). Infecting fibroblasts with adenovirus for dominant negative Rac1 (RAdN17rac1) had no marked effect on the induction of proMMP-13 production by TGF-ß1 compared to RAd66-infected cells. In accordance with the results obtained with p38 inhibitor SB203580, adenovirus-mediated expression of dominant negative p38 (RAdp38AF) markedly (by 76%) reduced induction of proMMP-13 production by TGF-ß1 compared with RAd66-infected cells (Fig. 2B ). Production of TIMP-1 was enhanced by TGF-ß1 in cells infected with RAd66 and RAdN17rac1 (2.8- and 1.6-fold, respectively), whereas adenovirus-mediated expression of dominant negative p38 slightly inhibited enhancement of TIMP-1 production by TGF-ß1 (Fig. 2B ). These results corroborate the observation that TGF-ß-elicited induction of MMP-13 expression in fetal skin fibroblasts is mediated by p38 MAPK.

CONCLUSIONS

We have previously noted that MMP-13 is expressed by fibroblasts during normal repair of human gingival wounds, characterized by minimal scar formation. In addition, MMP-13 is expressed by fibroblasts in chronic human cutaneous ulcers in vivo but not in normally healing dermal wounds. Here we show that MMP-13 is expressed by fibroblasts in acute wound repair of human fetal skin grafted on SCID mice. We also show that fetal skin fibroblasts in monolayer culture express MMP-13 and that the expression is induced by TGF-ß1 via the p38 MAPK signaling pathway. Expression of MMP-13 has been detected in human gingival fibroblasts treated with TGF-ß1. In contrast, the expression of MMP-13 in normal human skin fibroblasts in monolayer culture is undetectable, but can be induced by culturing them in 3-dimensional collagen. Thus, our results show an interesting phenotypic similarity between human gingival and fetal skin fibroblasts, both of which express MMP-13 during normal wound repair in vivo, and in culture, when exposed to TGF-ß1.

Our results show that, in contrast to human fetal skin fibroblasts, neonatal skin fibroblasts in culture resemble adult skin fibroblasts, as they do not express detectable levels of MMP-13. This suggests that the ability to express MMP-13 is characteristic for fetal skin fibroblasts and that the change in fibroblast collagenolytic phenotype occurs within a short period around the time of birth. Previous studies have also documented other differences between adult and fetal skin fibroblasts in culture. Fetal skin fibroblasts migrate more efficiently into 3-dimensional collagen gel than adult fibroblasts. In addition, fetal skin fibroblasts display a higher proliferation rate, and they express lower levels of urokinase-type plasminogen activator and MMP-1 compared with adult skin fibroblasts. Fetal skin fibroblasts express lower levels of fibroblast growth factors 1, -2, and TGF-ß1 and respond differently to TGF-ß1 compared with adult human skin fibroblasts. Specifically, TGF-ß1 inhibits migration and hyaluronan (HA) synthesis of fetal skin fibroblasts, but not of adult skin fibroblasts. In addition, migration and HA synthesis of confluent fetal fibroblasts is inhibited by all three TGF-ß isoforms, whereas the migration and HA synthesis of confluent adult fibroblasts are stimulated by TGF-ß3, but not by TGF-ß1 and -ß2.

The production of MMP-13 by fetal skin fibroblasts was induced by TGF-ß1, a growth factor involved in all phases of cutaneous wound repair. TGF-ß1 is a potent activator of monocyte chemotaxis, fibroblast proliferation, formation of granulation tissue, and angiogenesis. TGF-ß1 induces the expression of several matrix components in adult dermal fibroblasts in vitro and in vivo, and addition of TGF-ß1 into rabbit fetal excisional wounds increases fibrosis and type I collagen expression. However, in adult rat wounds, blocking the activity of TGF-ß1 and -2 relative to TGF-ß3 inhibits scar formation. The role of TGF-ß3 as a scar reducing growth factor remains unclear since, in the rabbit ear model, TGF-ß3 does not reduce scarring, and in human lung fibroblasts TGF-ß1 and TGF-ß3 are equally potent in increasing ECM deposition, decreasing MMP-1 secretion, and increasing TIMP-1 expression. Our results show that TGF-ß1 and TGF-ß3 have a similar effect on MMP-13 expression by fetal skin fibroblasts, suggesting that both play a role in the up-regulation of MMP-13 expression and rapid turnover of collagen during fetal wound repair.

In conclusion, the results of the present study show that the regulation of the collagenolytic capacity in human fetal skin fibroblasts is similar to that of human gingival fibroblasts. These observations also demonstrate a fundamental difference in the regulation of collagenolytic capacity between fetal and neonatal skin fibroblasts, especially in response to TGF-ß1, a growth factor implicated in ECM accumulation in wound repair and fibrosis. It is therefore possible that as an MMP with a wide substrate specificity, MMP-13 may play an important role in rapid turnover of granulation tissue ECM during fetal wound repair, resulting in minimal scar formation.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram showing the possible role of collagenase-3 (MMP-13) in human fetal skin wound repair. TGF-ß induces () production of MMP-13 by fetal skin fibroblasts during wound repair and enhances turnover of collagenous granulation tissue, resulting in minimal scar formation. In adult dermal fibroblasts, TGF-ß inhibits () production of collagenase-1 (MMP-1) and reduces turnover of granulation tissue resulting in wound repair with scar.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0588fje ; to cite this article, use FASEB J. (February 20, 2001) 10.1096/fj.00-0588fje

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