Role of MMP3 and fibroblast-MMP14 in skin homeostasis and repair.

Early lethality of mice with complete deletion of the matrix metalloproteinase MMP14 emphasized the proteases' pleiotropic functions. MMP14 deletion in adult dermal fibroblasts (MMP14Sf-/-) caused collagen type I accumulation and upregulation of MMP3 expression. To identify the compensatory role of MMP3, mice were generated with MMP3 deletion in addition to MMP14 loss in fibroblasts. These double deficient mice displayed a fibrotic phenotype in skin and tendons as detected in MMP14Sf-/- mice, but no additional obvious defects were detected. However, challenging the mice with full thickness excision wounds resulted in delayed closure of early wounds in the double deficient mice compared to wildtype and MMP14 single knockout controls. Over time wounds closed and epidermal integrity was restored. Interestingly, on day seven, post-wounding myofibroblast density was lower in the wounds of all knockout than in controls, they were higher on day 14. The delayed resolution of myofibroblasts from the granulation tissue is paralleled by reduced apoptosis of these cells, although proliferation of myofibroblasts is induced in the double deficient mice. Further analysis showed comparable TGFß1 and TGFßR1 expression among all genotypes. In addition, in vitro, fibroblasts lacking MMP3 and MMP14 retained their ability to differentiate into myofibroblasts in response to TGFß1 treatment and mechanical stress. However, in vivo, p-Smad2 was reduced in myofibroblasts at day 5 post-wounding, in double, but most significant in single knockout, indicating their involvement in TGFß1 activation. Thus, although MMP3 does not compensate for the lack of fibroblast-MMP14 in tissue homeostasis, simultaneous deletion of both proteases in fibroblasts delays wound closure during skin repair. Notably, single and double deficiency of these proteases modulates myofibroblast formation and resolution in wounds.

Loss of Endothelial Cell Matrix Metalloproteinase 14 Reduces Melanoma Growth and Metastasis by Increasing Tumor Vessel Stability.

Matrix metalloproteinase (MMP) 14 belongs to a large family of zinc-dependent endopeptidases and plays a critical role in skin physiological and pathological processes. Complete loss of the protease resulted in severe developmental defects leading to early death. However, because of the premature death of the mice, the functional significance for endothelial cell (EC) expression of MMP14 in skin physiology and pathology in vivo after birth is yet unknown. Using a mouse model with constitutive EC-specific deletion of Mmp14 (Mmp14EC‒/‒), we showed that mice developed and bred normal, but melanoma growth and metastasis were reduced. Although vascularity was unaltered, vessel permeability was decreased. Deletion of MMP14 in ECs led to increased vessel coverage by pericytes and vascular endothelial-cadherin expression in mice in vivo and in vitro but not in human ECs. Endothelial nitric oxide synthase expression and nitric oxide production were significantly reduced in Mmp14EC‒/‒ ECs and MMP14-silenced human umbilical vein ECs. A direct correlation between endothelial nitric oxide synthase and MMP14 expression was detected in intratumoral vessels of human malignant melanomas. Altogether, we show that endothelial MMP14 controls tumor vessel function during melanoma growth. These data suggest that EC-derived MMP14 direct targeting alone or with vascular stabilizing agents may be therapeutically crucial in inhibiting melanoma growth and metastasis.

Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth.

Maintaining a balanced state in remodeling the extracellular matrix is crucial for tissue homeostasis, and this process is altered during skin cancer progression. In melanoma, several proteolytic enzymes are expressed in a time and compartmentalized manner to support tumor progression by generating a permissive environment. One of these proteases is the matrix metalloproteinase 14 (MMP14). We could previously show that deletion of MMP14 in dermal fibroblasts results in the generation of a fibrotic-like skin in which melanoma growth is impaired. That was primarily due to collagen I accumulation due to lack of the collagenolytic activity of MMP14. However, as well as collagen I processing, MMP14 can also process several extracellular matrices. We investigated extracellular matrix alterations occurring in the MMP14-deleted fibroblasts that can contribute to the modulation of melanoma growth. The matrix deposited by cultured MMP14-deleted fibroblast displayed an antiproliferative and anti-migratory effect on melanoma cells in vitro. Analysis of the secreted and deposited-decellularized fibroblast's matrix identified a few altered proteins, among which the most significantly changed was collagen XIV. This collagen was increased because of post-translational events, while de novo synthesis was unchanged. Collagen XIV as a substrate was not pro-proliferative, pro-migratory, or adhesive, suggesting a negative regulatory role on melanoma cells. Consistent with that, increasing collagen XIV concentration in wild-type fibroblast-matrix led to reduced melanoma proliferation, migration, and adhesion. In support of its anti-tumor activity, enhanced accumulation of collagen XIV was detected in peritumoral areas of melanoma grown in mice with the fibroblast's deletion of MMP14. In advanced human melanoma samples, we detected reduced expression of collagen XIV compared to benign nevi, which showed a robust expression of this molecule around melanocytic nests. This study shows that loss of fibroblast-MMP14 affects melanoma growth through altering the peritumoral extracellular matrix (ECM) composition, with collagen XIV being a modulator of melanoma progression and a new proteolytic substrate to MMP14.

Epidermal NLRP10 contributes to contact hypersensitivity responses in mice.

The nucleotide binding and oligomerization domain-like receptor (NLR) protein NLRP10 is highly expressed in the epidermis and contributes to cell-autonomous responses against invasive bacteria. To investigate the role of NLRP10 in inflammatory responses of the skin we analyzed the effect of full-body and keratinocyte-specific depletion of NLRP10 in croton oil-induced irritant contact dermatitis (ICD) and 1-fluoro-2,4-dinitrobenzene (DNFB)-induced contact hypersensitivity (CHS) in mice. Nlrp10(-/-) mice were phenotypically normal and skin repair after wounding was not affected by lack of NLRP10. Similarly, we did not detect a contribution of NLRP10 to the ICD response induced by croton oil. In contrast, Nlrp10(-/-) mice showed significantly reduced inflammation in the DNFB-induced CHS response as compared to control animals. Microscopic analysis revealed significantly reduced numbers of CD4(+) and CD8(+) T cells in the infiltrates of animals lacking NLRP10 expression after CHS challenge. Epidermis-specific deletion of Nlrp10 by keratin-14 promotor driven Cre-recombinase was sufficient to account for this phenotype, although lymphocyte recruitment seemed to be unaltered in animals lacking NLRP10 expression in keratinocytes. Taken together, we provide evidence that NLRP10 contributes to T-cell-mediated inflammatory responses in the skin and highlight a physiological role of NLRP10 in epidermal keratinocytes.

Loss of epidermal MMP-14 expression interferes with angiogenesis but not with re-epithelialization.

Synthesis and activation of matrix metalloproteinases during wound healing are important for remodeling the extracellular matrix and modulating various cellular functions. The membrane-type 1 matrix metalloproteinase (MMP-14) has been shown to play a key role during these processes. To analyze the function of epidermal-derived MMP-14 during skin repair we generated mice lacking MMP-14 expression in the epidermis (MMP-14(ep-/-)). These mice displayed overall normal skin morphology and epidermal differentiation patterns. Wound repair in MMP-14(ep-/-) followed the same kinetics as in wild type mice (MMP-14(ep+/+)), and infiltration of neutrophils, leukocytes, and macrophages into the wound site was comparable. Microscopic analysis showed no altered re-epithelialization in the absence of epidermal MMP-14. Furthermore, epidermal differentiation at the end of the repair process and scar formation was normal. However, at day 14 post wounding, sustained angiogenesis was observed in MMP-14(ep-/-) mice in contrast to control mice. Interestingly, decreased levels of endostatin were detected in wound lysates of MMP-14(ep-/-) mice as well as in cultured keratinocytes. Taken together, these data indicate that MMP-14 expression in keratinocytes is dispensable for skin homeostasis and repair, but plays a crucial role in the epidermal-dermal crosstalk leading to modulation of vessel density.

Role of ADAM-15 in wound healing and melanoma development.

Proteins of the a disintegrin and metalloprotease (ADAM) family are transmembrane proteins involved in ectodomain shedding and in cellular interactions. In skin, ADAM-15 is detected in the epidermis and dermal vascular structures by immunolocalization. Expression is also detected in isolated fibroblast, keratinocytes and endothelial cells in culture. Despite high expression of ADAM-15 throughout the wound repair process, wound healing experiments in vivo revealed a dispensable role of ADAM-15 for the healing process. No alterations in wound closure, re-epithelialization, contraction, scar formation and angiogenesis were detected in animals carrying ADAM-15-/- deletion. When analysing melanoma development by grafting melanoma cells into the flank of ADAM-15-/-, no significant alteration in tumor growth was detected. However, at later stages, melanomas in the ADAM-15-/- animals were smaller than those grown in WT animals. At all time points, no significant differences in vascularization of the peritumoral stroma and tumors were detected. Interestingly, we could detect a reduced number of metastasized lungs and lymph nodes in ADAM-15-/- animals as compared to control littermate mice. In conclusion, our study indicated that ADAM-15 is dispensable for cutaneous wound healing and B16F1 melanoma growth, but significantly contributes to metastasis formation.

Stromal fibroblast-specific expression of ADAM-9 modulates proliferation and apoptosis in melanoma cells in vitro and in vivo.

ADAMs are members of the zinc metalloproteinase superfamily characterized by the presence of disintegrin and metalloprotease domains. In human melanoma, ADAM-9 is expressed in focalized areas of the tumor-stroma border in both melanoma and stromal cells. However, the role of ADAM-9 in melanoma progression remains elusive. To analyze the role of stromal-derived ADAM-9 for the growth and survival of melanoma cells, we have used in vitro coculture systems of melanoma cells and ADAM-9(-/-) fibroblasts. Coculture of melanoma cells in the presence of ADAM-9(-/-) fibroblasts led to increased melanoma cell proliferation and reduced apoptosis as compared with control cocultures. We identified TIMP-1 and sTNFRI as the two relevant factors expressed in increased amounts in culture supernatants from ADAM-9(-/-) fibroblasts. TIMP-1 was associated with induced melanoma cell proliferation, whereas soluble TNFR1 mediated the reduced cellular apoptosis in vitro. In vivo, injection of murine melanoma cells into the flank of ADAM-9(-/-) animals resulted in the development of significantly larger tumors than in wild-type animals as a result of increased proliferation and decreased apoptosis of melanoma cells. Taken together, stromal expression of ADAM-9 during melanoma development modulates the expression of TIMP-1 and sTNFR1, which in turn affect tumor cell proliferation and apoptosis.

Accelerated wound repair in ADAM-9 knockout animals.

ADAM-9 belongs to a family of transmembrane, disintegrin-containing metalloproteinases (ADAMs) involved in protein ectodomain shedding and cell-cell and cell-matrix interactions. Although the functions of many ADAM family members are known, the specific biological function of ADAM-9 is still unclear. In this study, we have analyzed ADAM-9 temporal and spatial distribution during wound healing. We showed increased ADAM-9 transcript expression during the first 7 days post-wounding and, by immunolocalization, detected ADAM-9 in all migrating and proliferating keratinocytes from days 3 to 7. In older 14-day-old wounds, ADAM-9 expression was restored. We have investigated the role of this protein in the healing process following excisional wounding. Animals deficient in ADAM-9 showed accelerated wound repair compared with control littermates. No alterations in neutrophil, leukocyte, and macrophage infiltration were observed. However, re-epithelialization was significantly faster in Adam-9 -/- than control wounds. Although no differences in proliferation were observed in vivo and in vitro, increased migration of keratinocytes was responsible for this effect. These results show the previously unreported role of ADAM-9 in wound repair by regulating keratinocyte migration through modulation of collagen XVII shedding.

Stromal expression of MMP-13 is required for melanoma invasion and metastasis.

Tumor invasion and metastasis of malignant melanoma have been shown to require proteolytic degradation of the extracellular environment achieved primarily by enzymes of the matrix metalloproteinases (MMP) family. We have earlier shown that increased enzyme activity is localized at the border of tumor cells and the adjacent peritumoral connective tissue, emphasizing the importance of tumor-stroma interactions in the regulation of MMP activity. To confirm the role of stroma-derived MMP-13 in the invasion process, we investigated the invasiveness of melanoma cells upon intradermal injection in mice with complete inactivation of MMP-13. Tumor growth was significantly impaired in mmp-13(-/-) mice and most significant at early time points as compared with wild-type littermates. Moreover, metastasis to various organs was reduced to 17.6 vs 30% in lungs, 2.9 vs 30% in the liver. Strikingly, ablation of MMP-13 completely abrogated formation of metastasis in the heart (0 vs 40%). Notably, decreased tumor growth in mmp-13(-/-) mice was associated with reduced blood vessel density. In addition, decreased blood vessel permeability in the tumors was measured by magnetic resonance imaging of tumor-bearing animals. These data suggest an important role of MMP-13 in tumor growth and an unexpected role in organ-specific metastasis of melanoma cells.

Identification of susceptibility loci for skin disease in a murine psoriasis model.

Psoriasis is a frequently occurring inflammatory skin disease characterized by thickened erythematous skin that is covered with silvery scales. It is a complex genetic disease with both heritable and environmental factors contributing to onset and severity. The CD18 hypomorphic PL/J mouse reveals reduced expression of the common chain of beta(2) integrins (CD11/CD18) and spontaneously develops a skin disease that closely resembles human psoriasis. In contrast, CD18 hypomorphic C57BL/6J mice do not demonstrate this phenotype. In this study, we have performed a genome-wide scan to identify loci involved in psoriasiform dermatitis under the condition of low CD18 expression. Backcross analysis of a segregating cross between susceptible CD18 hypomorphic PL/J mice and the resistant CD18 hypomorphic C57BL/6J strain was performed. A genome-wide linkage analysis of 94 phenotypically extreme mice of the backcross was undertaken. Thereafter, a complementary analysis of the regions of interest from the genome-wide screen was done using higher marker density and further mice. We found two loci on chromosome 10 that were significantly linked to the disease and interacted in an additive fashion in its development. In addition, a locus on chromosome 6 that promoted earlier onset of the disease was identified in the most severely affected mice. For the first time, we have identified genetic regions associated with psoriasis in a mouse model resembling human psoriasis. The identification of gene regions associated with psoriasis in this mouse model might contribute to the understanding of genetic causes of psoriasis in patients and pathological mechanisms involved in development of disease.

CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.

In a CD18 hypomorphic polygenic PL/J mouse model, the severe reduction of CD18 (beta(2) integrin) to 2-16% of wild-type levels leads to the development of a psoriasiform skin disease. In this study, we analyzed the influence of reduced CD18 gene expression on T cell function, and its contribution to the pathogenesis of this disease. Both CD4(+) and CD8(+) T cells were significantly increased in the skin of affected CD18 hypomorphic mice. But only depletion of CD4(+) T cells, and not the removal of CD8(+) T cells, resulted in a complete clearance of the psoriasiform dermatitis. This indicates a central role of CD4(+) T cells in the pathogenesis of this disorder, further supported by the detection of several Th1-like cytokines released predominantly by CD4(+) T cells. In contrast to the CD18 hypomorphic mice, CD18 null mutants of the same strain did not develop the psoriasiform dermatitis. This is in part due to a lack of T cell emigration from dermal blood vessels, as experimental allergic contact dermatitis could be induced in CD18 hypomorphic and wild-type mice, but not in CD18 null mutants. Hence, 2-16% of CD18 gene expression is obviously sufficient for T cell emigration driving the inflammatory phenotype in CD18 hypomorphic mice. Our data suggest that the pathogenic involvement of CD4(+) T cells depends on a gene dose effect with a reduced expression of the CD18 protein in PL/J mice. This murine inflammatory skin model may also have relevance for human polygenic inflammatory diseases.