V. Martins

Matrix metalloproteinases and epidermal wound repair.

Epidermal wound healing is a complex and highly coordinated process where several different cell types and molecules, such as growth factors and extracellular matrix (ECM) components, play an important role. Among the many proteins that are essential for the restoration of tissue integrity is the metalloproteinase (MMP) family. MMPs can act on ECM and non-ECM components affecting degradation and modulation of the ECM, growth-factor activation and cell–cell and cell–matrix signalling. MMPs are secreted by different cell types such as keratinocytes, fibroblasts and inflammatory cells at different stages and locations during wound healing, thereby regulating this process in a very coordinated and controlled way. In this article, we review the role of MMPs and their inhibitors (TIMPs), as well as the disintegrin and metalloproteinase with the thrombospondin motifs (ADAMs) family, in epithelial wound repair.

Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of basement-membrane type VII collagen

Type VII collagen (ColVII) is the main component of anchoring fibrils, attachment structures within the lamina densa of the basement membrane that are responsible for attachment of the epidermis to the dermis in skin. Mutations in the human ColVII gene, COL7A1, cause the severe inherited blistering disorder recessive dystrophic epidermolysis bullosa (RDEB) affecting skin and mucosae, associated with a greatly increased risk of skin cancer. In this study, we examined the effect of loss of ColVII on squamous cell carcinoma (SCC) tumourigenesis using RNAi in a 3D organotypic skin model. Our findings suggest that loss of ColVII promotes SCC migration and invasion as well as regulating cell differentiation with evidence for concomitant promotion of epithelial-mesenchymal transition (EMT). Immunostaining of RDEB skin and a tissue array of sporadic cutaneous SCCs confirmed that loss of ColVII correlates with decreased involucrin expression in vivo. Gene-expression-array data and immunostaining demonstrated that loss of ColVII increases expression of the chemokine ligand-receptor CXCL10-CXCR3 and downstream-associated PLC signalling, which might contribute to the increased metastatic potential of SCCs with reduced or absent ColVII expression. Together, these findings may explain the aggressive behaviour of SCCs in RDEB patients and may also be relevant to non-RDEB skin cancer, as well as other tumours from organs where ColVII is expressed.

Suppression of TGFβ and Angiogenesis by Type VII Collagen in Cutaneous SCC

BACKGROUND Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGFβ signaling and angiogenesis in vitro and in vivo. METHODS Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGFβ pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided. RESULTS Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between α2β1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFβ signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFβ markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFβ markers, n = 6; VEGF, n = 21). Inhibition of TGFβ receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siTβRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS Type VII collagen suppresses TGFβ signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.

Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of type VII collagen

Collagen XVII (COL17), a transmembrane collagen, is thought to be involved in keratinocyte adhesion and possibly migration, as COL17 defects disrupt keratinocytebasal lamina adhesion and underlie the disease non-Herlitz junctional epidermolysis bullosa (n-HJEB). COL17 is involved in keratinocyte adhesion and migration. Using siRNA to knockdown COL17 expression in HaCaT cells, we assessed cell characteristics including adhesion, migration and signaling. Control and siRNA transfected keratinocytes showed no difference in adhesion on plastic dishes after incubation for 8 hours in serum-free keratinocyte-growth medium, however when grown on collagen IV alone or BD matrigel (containing collagen IV and laminin isoforms) COL17 defi cient cells showed signifi cantly reduced adhesion compared to controls (P<0.01), and MEK1/2 and MAPK demonstrated reduced phosphorylation in COL17 depleted cells. Furthermore, COL17 defi cient HaCaT cells plated on plastic exhibited reduced motility that was p38MAP kinase dependent (after addition of the p38MAPK inhibitor SB203580). Conversely, keratinocyte adhesion was independent of p38MAPK signaling. Taken together, these results suggest COL17 has signifi cantly wider signaling roles than were previously thought, including the involvement of COL17 in keratinocyte adhesion to collagen IV, in p38MAP kinase-dependent cell migration, and multiple cell signaling events pertaining to MEK1/2 phosphorylation.