Proteomic Analysis of Dupuytren's Contracture-Derived Sweat Glands Revealed the Synthesis of Connective Tissue Growth Factor and Initiation of Epithelial-Mesenchymal Transition as Major Pathogenetic Events.

Dupuytren's contracture (DC) is a chronic and progressive fibroproliferative disorder restricted to the palmar fascia of the hands. Previously, we discovered the presence of high levels of connective tissue growth factor in sweat glands in the vicinity of DC nodules and hypothesized that sweat glands have an important role in the formation of DC lesions. Here, we shed light on the role of sweat glands in the DC pathogenesis by proteomic analysis and immunofluorescence microscopy. We demonstrated that a fraction of sweat gland epithelium underwent epithelial-mesenchymal transition illustrated by negative regulation of E-cadherin. We hypothesized that the increase in connective tissue growth factor expression in DC sweat glands has both autocrine and paracrine effects in sustaining the DC formation and inducing pathological changes in DC-associated sweat glands.

Stimulation with THBS4 activates pathways that regulate proliferation, migration and inflammation in primary human keratinocytes.

As in other mammalian tissues, the extracellular matrix (ECM) of skin functions as mechanical support and regulative environment that guides the behavior of the cells. ECM is a gel-like structure that is primarily composed of structural and nonstructural proteins. While the content of structural proteins is stable, the level of nonstructural ECM proteins, such as thrombospondin-4 (THBS4), is dynamically regulated. In a previous work we demonstrated that THBS4 stimulated cutaneous wound healing. In this work we discovered that in addition to proliferation, THBS4 stimulated the migration of primary keratinocytes in 3D. By using a proteotransciptomic approach we found that stimulation of keratinocytes with THBS4 regulated the activity of signaling pathways linked to proliferation, migration, inflammation and differentiation. Interestingly, some of the regulated genes (eg IL37, TSLP) have been associated with the pathogenesis of atopic dermatitis (AD). We concluded that THBS4 is a promising candidate for novel wound healing therapies and suggest that there is a potential convergence of pathways that stimulate cutaneous wound healing with those active in the pathogenesis of inflammatory skin diseases.

Matricellular proteins in cutaneous wound healing.

Cutaneous wound healing is a complex process that encompasses alterations in all aspects of the skin including the extracellular matrix (ECM). ECM consist of large structural proteins such as collagens and elastin as well as smaller proteins with mainly regulative properties called matricellular proteins. Matricellular proteins bind to structural proteins and their functions include but are not limited to interaction with cell surface receptors, cytokines, or protease and evoking a cellular response. The signaling initiated by matricellular proteins modulates differentiation and proliferation of cells having an impact on the tissue regeneration. In this review we give an overview of the matricellular proteins that have been found to be involved in cutaneous wound healing and summarize the information known to date about their functions in this process.

Olfactomedin 4 regulates migration and proliferation of immortalized non-transformed keratinocytes through modulation of the cell cycle machinery and actin cytoskeleton remodelling.

Olfactomedin 4 (OLFM4), a multifunctional matricellular protein, is involved in regulation of angiogenesis, innate immunity, inflammation, tumorigenesis and metastasis formation via modulation of important cellular processes like adhesion, proliferation, differentiation as well as apoptosis. In our previous work we demonstrated the upregulation of OLFM4 during liver regeneration and cutaneous wound healing. Here we studied the outcomes of OLFM4 downregulation in human immortalized keratinocytes - the HaCaT cells. The suppression of OLFM4 inhibited migration but enhanced the proliferation of these cells. By using proteomic, and phosphoproteomic analysis, we found that OLFM4 downregulation induced changes in the levels of 184 proteins and 348 phosphosites. An integrated pathway analysis suggested that the increased phosphorylation of CDK7 at Ser164 and Thr170 may serve as the key event in the activation of CDK2 and consequent activation of cell cycle progression. Furthermore, the decrease in GIT1 and WAVE2 protein levels were connected to the disorganization of the actin cytoskeleton, reduction of lamellipodia formation at the leading edge of HaCaT cells, and decrease in their migration capacity.

Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing.

Thrombospondin-4 (THBS4) is a non-structural extracellular matrix molecule associated with tissue regeneration and a variety of pathological processes characterized by increased cell proliferation and migration. However, the mechanisms of how THBS4 regulates cell behavior as well as the pathways contributing to its effects have remained largely unexplored. In the present study we investigated the role of THBS4 in skin regeneration both in vitro and in vivo. We found that THBS4 expression was upregulated in the dermal compartment of healing skin wounds in humans as well as in mice. Application of recombinant THBS4 protein promoted cutaneous wound healing in mice and selectively stimulated migration of primary fibroblasts as well as proliferation of keratinocytes in vitro. By using a combined proteotranscriptomic pathway analysis approach we discovered that ß-catenin acted as a hub for THBS4-dependent cell signaling and likely plays a key role in promoting its downstream effects. Our results suggest that THBS4 is an important contributor to wound healing and its incorporation into novel wound healing therapies may be a promising strategy for treatment of cutaneous wounds.