Keratinocyte Collagenase-1 Expression Requires an Epidermal Growth Factor Receptor Autocrine Mechanism (original) (raw)
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Journal of Clinical Investigation, 1993
We reported that interstitial collagenase is produced by keratinocytes at the edge of ulcers in pyogenic granuloma, and in this report, we assessed if production of this metalloproteinase is a common feature of the epidermal response in a variety of wounds. In all samples of chronic ulcers, regardless of etiology, and in incision wounds, collagenase mRNA, localized by in situ hybridization, was prominently expressed by basal keratinocytes bordering the sites ofactive re-epithelialization indicating that collagenolytic activity is a characteristic response of the epidermis to wounding. No expression of mRNAs for 72-and 92-kD gelatinases or matrilysin was seen in keratinocytes, and no signal for any metalloproteinase was detected in normal epidermis. Immunostaining for type IV collagen showed that collagenase-positive keratinocytes were not in contact with an intact basement membrane and, unlike normal keratinocytes, expressed a5jh receptors. These observations suggest that cellmatrix interactions influence collagenase expression by epidermal cells. Indeed, as determined by ELISA, primary cultures of human keratinocytes grown on basement membrane proteins (Matrigel; Collaborative Research Inc., Bedford, MA) did not express significant levels of collagenase, whereas cells grown on type I collagen produced markedly increased levels. These results suggest that migrating keratinocytes actively involved in re-epithelialization acquire a collagenolytic phenotype upon contact with the dermal matrix.
British Journal of Dermatology, 2006
Background Hypertrophic scarring and skin graft contracture are major causes of morbidity after burn injuries. A prominent feature is excessive fibroplasia with accumulation of increased fibrillar collagen relative to normal scar tissue. The application of split-thickness skin grafts or cultured epithelial autografts to burn wounds is known to reduce scarring and contraction. Objectives To investigate further how the keratinocyte influences underlying fibroblast behaviour by examining the influence of keratinocytes on fibroblast collagen synthesis, using a new assay for collagen synthesis never previously applied to skin cell biology. Methods We investigated the influence of the keratinocyte on fibroblast synthesis of type I collagen using an immunoassay for the aminoterminal propeptide of type I collagen (P1NP) in conditioned medium from monocultures and cocultures of keratinocytes and fibroblasts over 14 days. The importance of the physical presence of the keratinocyte was investigated by comparing cocultures of keratinocytes and fibroblasts against fibroblast monocultures with keratinocyteconditioned medium. Pharmacological agents known to promote fibroblast proliferation [basic fibroblast growth factor (bFGF)], keratinocyte proliferation [insulin-like growth factor (IGF)-1], modify scarring in vivo [tumour necrosis factor (TNF)-a] or modify collagen biochemistry [putrescine, estrone, estradiol and b-aminopropionitrile (b-APN)] were then investigated for their effect on collagen synthesis in fibroblasts and in keratinocyte ⁄fibroblast cocultures. Results Keratinocytes in coculture with fibroblasts, and keratinocyte-conditioned medium, both reduced fibroblast P1NP synthesis. Of the pharmacological agents investigated, bFGF, IGF-1, TNF-a and b-APN all increased collagen synthesis both in monocultures of fibroblasts and in cocultures of keratinocytes and fibroblasts. Conclusions Fibroblast collagen synthesis appears to be downregulated by keratinocyte-derived cytokines. Fibroblast growth factors and proinflammatory cytokines appear to be able partially to overcome this downregulation and to increase collagen synthesis.
Matrix Biology, 2006
EGF and type I collagen are known to play important roles in wound healing. In the present study, we demonstrated that EGF down-regulates the expression of type I procollagen protein as well as a2(I) collagen mRNA in cultured human dermal fibroblasts. EGF induced the degradation of type I procollagen protein in conditioned medium through the up-regulation of MMP-1 expression. EGF down-regulated a2(I) mRNA expression partially at the post-transcriptional level by reducing the mRNA stability. In contrast, EGF up-regulated MMP-1 mRNA expression mostly at the transcriptional level, in that it had a stimulatory effect on MMP-1 promoter activity, but no effect on MMP-1 mRNA stability. The MEK/ERK signaling pathway was shown to be involved in EGF-mediated type I collagen and MMP-1 expression.
Keratinocyte-conditioned media regulate collagen expression in dermal fibroblasts
Journal of Investigative Dermatology, 2008
Excessive extracellular matrix (ECM) production during dermal wound healing often leads to fibrotic conditions such as keloids and hypertrophic scarring (HSc). Type I collagen is the predominant form of collagen in the human skin and is produced mainly by dermal fibroblasts. It has been suggested that abnormalities in epidermal-dermal interaction can lead to excessive production of collagen by fibroblasts. To identify and further characterize any possible keratinocyte-derived collagen-inhibitory factors (KD-CIFs), we investigated the expression of pro-a1(I) collagen at the level of mRNA and protein in human fibroblasts that had been either cocultured with keratinocytes or treated with keratinocyte-conditioned medium (KCM). Fibroblasts in both groups demonstrated a significant reduction in the steady-state levels of collagen mRNA and protein. Further characterization of KD-CIFs revealed a high-molecular-weight factor (430 kDa) that showed stable activity at high temperature (561C) and acidic pH (pH 2). Keratinocyte differentiation did not alter the release of KD-CIFs into KCM. These results provide further evidence that type I collagen expression and synthesis in fibroblasts are regulated by a keratinocyte-releasable factor(s) with an apparent molecular weight between 30 and 50 kDa.
British Journal of Dermatology, 1995
We have previously shown that conditioned medium from cultured human keratinocytes stimulates proliferation of a variety of cell types involved in wound heaiing. as well as re-epithelialization of wounds in human skin in vitro. We now present evidence for an autocrine/paracrine control ofthe synthesis of type IV collagenases in human keratinocytes and tihrohlasts. During wound healing, keratinocytes migrate over the wound hed. an activity coupled with lysis of hasement membranes, and hence requiring the presence of collagenases. Collagenases are also needed for the production and remodelling of the granulation tissue. In order to study the autocrine/paracrine control of collagenase production in keratinocy:es and fibroblasts. we stimulated these cells in culture with conditioned medium from cultured keratinocytes. Protease synthesis was determined by affinity labelling with 'H-diisopropyllluorophosphoridate (DFP) and by zymography. Keratinocyte-conditioned medium was found to increase the expression of 72 and 92 kDa type IV coliagenase in human keratinocytes, and the 72 kDa collagenase in human tibroblasts. indicating that an autocrine/ paracrine control mechanism is invoked in collagenase production in these cell types during wound healing. This increased expression of collagenases could he partly responsible for the stimulated healing seen in wounds treated with sheets of cultured keratinocytes.
The Journal of biological chemistry, 1994
We have previously shown that during wound healing migrating keratinocytes, which are in contact with the dermal matrix, express interstitial collagenase, whereas basal epidermal cells, which reside on an intact basement membrane, do not. Duplicating this in vivo pattern, collagenase production was induced in primary human keratinocytes grown on native type I collagen, but only background levels of enzyme were detected in cells cultured on denatured type I collagen or on Matrigel. Using genistein, herbimycin A, and sodium orthovanadate, we show that tyrosine kinase activity was required for collagen-mediated induction of keratinocyte collagenase. Similarly, collagenase steady-state mRNA levels and the activity of a transfected human collagenase-promoter CAT construct were inhibited by genistein and enhanced by orthovanadate. Staurosporine and H-7 also blocked collagenase production, indicating that protein kinase C activity was also required for collagen-mediated induction of kerati...
Genetically Modified Dermal Keratinocytes Express High Levels of Transforming Growth Factor‐β1
Journal of Investigative Dermatology, 1998
The purpose of this study was to examine the appearance and activation of collagenase-1 (MMP-1) in the wound environment. We found that MMP-1 accumulates in the fluid phase of the burn wound environment within 2 d of injury and reaches maximal levels by day 4. Two forms of the enzyme were evident; one that corresponded to proMMP-1 and another that corresponded to a group of high molecular mass (µ200 kDa and >200 kDa doublet) MMP-1 containing complexes. ProMMP-1 and MMP-1 containing complexes also occurred in wound fluid from venous stasis ulcers, but neither was detected in mastec-