Hedgehog Signaling in Papillary Fibroblasts Is Essential for Hair Follicle Regeneration during Wound Healing.

Patients suffering from large scars such as burn victims not only encounter aesthetic challenges but also ongoing itching or pain that substantially deteriorates their quality of life. Skin appendages such as hair follicles rarely regenerate within the healing wound. Because they are crucial for skin homeostasis and the lack thereof constitutes one of the main limitations to scarless wound healing, their regeneration represents a major objective for regenerative medicine. Fibroblasts, the main resident cell type of the skin dermis, mediate embryonic hair follicle morphogenesis and are particularly involved in wound healing because they orchestrate extracellular matrix remodeling and collagen deposition in the wound bed. Importantly, dermal fibroblasts originate from two distinct developmental lineages with unique functions that differently mediate the response to epidermal signals such as Hedgehog signaling. In this study, we show that Hedgehog signaling in the reticular fibroblast lineage promotes the initial phase of wound repair, possibly by modulating angiogenesis and fibroblast proliferation, whereas Hedgehog signaling in papillary fibroblasts is essential to induce de novo hair follicle formation within the healing wound.

Inhibition of p38/MK2 Signaling Prevents Vascular Invasion of Melanoma.

Melanoma cells can switch between distinct gene expression profiles, resulting in proliferative or invasive phenotypes. Signaling pathways involved in this switch were analyzed by gene expression profiling of a cohort of 22 patient-derived melanoma cell lines. CDH1 negativity was identified as a surrogate marker for the invasive phenotype. CDH1 expression could be turned on and off by modulating activity of p38 or its downstream target MK2, suggesting that this pathway controls melanoma progression. Mechanistically, MK2 inhibition prevented melanoma-induced vascular barrier disruption, reduced the expression of PODXL and DEL-1, and prevented vascular dissemination in vivo. PODXL and DEL-1 expression in patients with melanoma were associated with poor survival and thus can be used as prognostic markers. Downstream targets of MK2 may thus serve as candidate therapeutics.

CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma.

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils in vivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. In vitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.

CXCL5 Facilitates Melanoma Cell-Neutrophil Interaction and Lymph Node Metastasis.

Chemokines influence tumor metastasis by targeting tumor, stromal, and hematopoietic cells. Characterizing the chemokine mRNA expression profile of human primary melanoma samples, we found CXCL5 significantly up-regulated in stage T4 primary melanomas when compared to thin melanomas (T1 stage). To characterize the role of CXCL5 in melanoma progression, we established a metastasizing murine xenograft model using CXCL5-overexpressing human melanoma cells. CXCL5 had no effect on melanoma proliferation in vitro and on primary tumor growth in vivo, but CXCL5-overexpressing tumors recruited high amounts of neutrophils and exhibited significantly increased lymphangiogenesis in our severe combined immune-deficient mouse model. Recruited neutrophils were found in close proximity to or within lymphatic vessels, often in direct contact with melanoma cells. Clinically, CXCL5-overexpressing melanomas had significantly increased lymph node metastases. We were able to translate these findings to human patient samples and found a positive correlation between CXCL5 expression, numbers of neutrophils in stage T4 primary melanoma, and the occurrence of subsequent locoregional metastasis.

Description of a Putative Oligosaccharyl:S-Layer Protein Transferase from the Tyrosine O-Glycosylation System of Paenibacillus alvei CCM 2051T.

Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacterium Paenibacillus alvei CCM 2051T is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the P. alvei S-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosine O-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present in P. alvei CCM 2051T, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic of O-oligosaccharyl:protein transferases (O-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy_C motif resulted in loss of WsfB function evidenced in reconstitution experiments in P. alvei ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in Escherichia coli CWG702 and Salmonella enterica SL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterial O-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase of P. alvei, and a promising candidate for the first O-OTase reported in Gram-positives.