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.

Fibroblast MMP14-Dependent Collagen Processing Is Necessary for Melanoma Growth.

Skin homeostasis results from balanced synthesis and degradation of the extracellular matrix in the dermis. Deletion of the proteolytic enzyme MMP14 in dermal fibroblasts (MMP14Sf-/-) leads to a fibrotic skin phenotype with the accumulation of collagen type I, resulting from impaired proteolysis. Here, we show that melanoma growth in these mouse fibrotic dermal samples was decreased, paralleled by reduced tumor cell proliferation and vessel density. Using atomic force microscopy, we found increased peritumoral matrix stiffness of early but not late melanomas in the absence of fibroblast-derived MMP14. However, total collagen levels were increased at late melanoma stages in MMP14Sf-/- mice compared to controls. In ex vivo invasion assays, melanoma cells formed smaller tumor islands in MMP14Sf-/- skin, indicating that MMP14-dependent matrix accumulation regulates tumor growth. In line with these data, in vitro melanoma cell growth was inhibited in high collagen 3D spheroids or stiff substrates. Most importantly, in vivo induction of fibrosis using bleomycin reduced melanoma tumor growth. In summary, we show that MMP14 expression in stromal fibroblasts regulates melanoma tumor progression by modifying the peritumoral matrix and point to collagen accumulation as a negative regulator of melanoma.

Inhibition of clathrin-mediated endocytosis by knockdown of AP-2 leads to alterations in the plasma membrane proteome.

In eukaryotic cells, clathrin-mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin-coated vesicles (CCVs) is AP-2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP-2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal-lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti-cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP-2 knockdown attenuated the global endocytic capacity, but clathrin-independent entry pathways were still operating, as indicated by persistent internalization of specific membrane-spanning and GPI-anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP-2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.

Loss of ADAM9 Leads to Modifications of the Extracellular Matrix Modulating Tumor Growth.

ADAM9 is a metalloproteinase strongly expressed at the tumor-stroma border by both tumor and stromal cells. We previously showed that the host deletion of ADAM9 leads to enhanced growth of grafted B16F1 melanoma cells by a mechanism mediated by TIMP1 and the TNF-α/sTNFR1 pathway. This study aimed to dissect the structural modifications in the tumor microenvironment due to the stromal expression of ADAM9 during melanoma progression. We performed proteomic analysis of peritumoral areas of ADAM9 deleted mice and identified the altered expression of several matrix proteins. These include decorin, collagen type XIV, fibronectin, and collagen type I. Analysis of these matrices in the matrix producing cells of the dermis, fibroblasts, showed that ADAM9-/- and wild type fibroblasts synthesize and secreted almost comparable amounts of decorin. Conversely, collagen type I expression was moderately, but not significantly, decreased at the transcriptional level, and the protein increased in ADAM9-/- fibroblast mono- and co-cultures with melanoma media. We show here for the first time that ADAM9 can release a collagen fragment. Still, it is not able to degrade collagen type I. However, the deletion of ADAM9 in fibroblasts resulted in reduced MMP-13 and -14 expression that may account for the reduced processing of collagen type I. Altogether, the data show that the ablation of ADAM9 in the host leads to the altered expression of peritumoral extracellular matrix proteins that generate a more favorable environment for melanoma cell growth. These data underscore the suppressive role of stromal expression of ADAM9 in tumor growth and call for a better understanding of how protease activities function in a cellular context for improved targeting.

Newly designed and validated impedance spectroscopy setup in microtiter plates successfully monitors viable biomass online.

In microtiter plates, conventional online monitoring of biomass concentration based on optical measurements is limited to transparent media: It also cannot differentiate between dead or viable biomass or suspended particles. To address this limitation, this study introduces and validates a new online monitoring setup based on impedance spectroscopy for detecting only viable biomass in 48- and 96-well microtiter plates. The setup was first validated electronically and characterized by determining the cell constants of the measuring geometry. Defined cell suspensions of Ustilago maydis, Hansenula polymorpha, Escherichia coli and Bacillus licheniformis were characterized to find, among other parameters, the most suitable frequency range and the characteristic frequency of ß-dispersion for each organism. Finally, the setup was exemplarily applied to monitor the growth of Hansenula polymorpha online. As reference, three different parallel cultures were performed in established cultivation systems. This new online monitoring setup based on impedance spectroscopy is robust and enables precise measurements of microbial biomass concentration. It is promising for future high-throughput applications.