Described as secreted and membrane-bound proteins important for neural pathfinding, the class of proteins called Semaphorins are expressed in multiple tissue types and are involved in diverse biologic processes. In this study, we describe the function of Semaphorin 7a, a membrane-bound Semaphorin known to stimulate neurite outgrowth, on human melanocytes. We show that Semaphorin 7a is expressed by human keratinocytes and fibroblasts in vitro and in vivo and that melanocytes express Plexin C1, a receptor for Semaphorin 7a. Upregulation of Semaphorin 7a was observed in fibroblasts treated with UV irradiation, a potent stimulus for melanocyte dendricity. Because of the importance of melanocyte dendrites in cutaneous photoprotection, we performed functional studies examining the effect of Semaphorin 7a in melanocyte dendrite formation. We also examined the contribution of beta1-integrin and Plexin C1 receptor signaling in mediating effects of Semaphorin 7a in melanocytes. We show that Semaphorin 7a induces significant melanocyte spreading and dendricity in human melanocytes. Furthermore, we show that beta1-integrins and Plexin C1 receptors are ligands for Semaphorin 7a, and that signaling by these receptors has opposing effects on Semaphorin 7a-induced dendrite formation.

Melanocytes are progenitor cells for melanoma, which arises through step-wise progression from dysplastic to invasive, to metastatic tumor. Our previous data showed that semaphorin 7A (Sema7A), a protein involved in axon guidance, stimulates melanocyte adhesion and dendricity through opposing actions of beta1-integrin and Plexin C1 receptors. We now show that Plexin C1 is diminished or absent in human melanoma cell lines; analysis of tissue microarrays of nevi, melanoma, and metastatic melanoma showed a decrease in Plexin C1 expression in metastatic melanoma, and an inverse correlation of Plexin C1 expression with depth of invasion. We examined the signaling intermediates of Sema7A and downstream targets of Plexin C1 in human melanocytes. Sema7A activated mitogen-activated protein kinase and inactivated cofilin, an actin-binding protein involved in cell migration. When Plexin C1 expression was silenced, Sema7A failed to phosphorylate cofilin, indicating that cofilin is downstream of Plexin C1. Further, Lim kinase II, a protein that phosphorylates cofilin, is upregulated by Sema7A in a Plexin C1-dependent manner. These data identify Plexin C1 as a potential tumor suppressor protein in melanoma progression, and suggest that loss of Plexin C1 expression may promote melanoma invasion and metastasis through loss of inhibitory signaling on cofilin activation.

Melanoma arises through complex genetic and epigenetic changes resulting in uncontrolled proliferation, invasion and metastatic disease. Semaphorins are critical regulators of axon guidance through interaction with their receptors, Plexins and neuropilins. Aberrant expression of Plexin receptors is been linked with progression of a variety of tumors. Plexin B1, the Semaphorin 4D receptor, activates the oncogenic receptors c-Met and ErbB-2, leading to speculation that it promotes tumor growth through stimulation of these receptors. We show that Plexin B1 is lost in metastatic melanoma and in deeply invasive primary tumors in vivo. Introduction of Plexin B1 into a human metastatic melanoma cell line suppressed proliferation, enhanced migration, stimulated Akt activation, and rendered cells resistant to cis-platin induced apoptosis. Unexpectedly, Plexin B1 inhibited c-Met by up to 54% in response to the c-Met ligand, hepatocyte growth factor, with a concordant loss of phosphorylation of the c-Met substrate, Gab1. Loss of Plexin B1 is predicted to function as a classic tumor suppressor protein in melanomas in which progression is c-Met dependent. However, because Plexin B1 activates Akt, and suppresses apoptosis, Plexin B1 likely plays a complex role in melanoma progression, and may be a predictive marker for tumor sensitivity to chemotherapeutic agents.

Melanocyte proliferation, dendrite formation, and pigmentation are controlled by paracrine factors, particularly following exposure to ultraviolet radiation (UVR). Little is known about autocrine factors for melanocytes. Prostaglandins activate signaling pathways involved in growth, differentiation and apoptosis. Prostaglandin E2 (PGE2) is the most abundant prostaglandin released by keratinocytes following UVR, and stimulates the formation of dendrites in melanocytes. Synthesis of PGE2 is controlled by cPLA2, which releases arachidonic acid from membranes, and COX-2 and prostaglandin E2 synthases (PGES), which convert arachidonic acid to PGH2 and PGH2 to PGE2, respectively. In this report we show that multiple irradiations of human melanocytes with UVR stimulates tyrosinase activity, independent of expression of a functional melanocortin 1 receptor, suggesting the presence of a non-melanocortin autocrine factor. Irradiation of melanocytes activated cPLA2, the rate-limiting step in eicosanoid synthesis, and stimulated PGE2 secretion. PGE2 increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE2 binds to four distinct G-protein coupled receptors (EP1–4). We show that EP4 receptor signaling stimulates cAMP production in melanocytes. Conversely, stimulation of the EP3 receptor lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE2 on cAMP in melanocytes. The data are the first to identify PGE2 as an UVR-inducible autocrine factor for melanocytes that stimulates tyrosinase activity and proliferation, and to show that EP3 and EP4 receptor signaling have opposing effects on cAMP production, a critical signaling pathway that regulates proliferation and melanogenesis in melanocytes.

Background: Posited pathological mechanisms in Facioscapulohumeral Muscular Dystrophy (FSHD) include activation in somatic tissue of normally silenced genes, increased susceptibility to oxidative stress, and induction of apoptosis. Objective: To determine the histopathological changes in FSHD muscle biopsies and compare to possible pathological mechanisms of disease. Methods: We performed a cross-sectional study on quadriceps muscle biopsies from 32 genetically confirmed FSHD participants, compared to healthy volunteers and myotonic dystrophy type 1 as disease controls. Biopsies were divided into groups to evaluate apoptosis rates, capillary density, myonuclear and satellite cell counts. Results: Apoptosis rates were increased in FSHD (n = 10, 0.74% ) compared to myotonic dystrophy type 1 (n = 10, 0.14% , P = 0.003) and healthy volunteers (n = 14, 0.13% , P = 0.002). Apoptosis was higher in FSHD patients with the smallest residual D4Z4 fragments. Capillary density was decreased in FSHD1 (n = 10, 316 capillaries/mm2) compared to healthy volunteers (n = 15, 448 capillaries/mm2, P = 0.001). No differences were seen in myonuclear or satellite cell counts. Conclusions: Preliminary evidence for increased apoptosis rates and reduced capillary density may reflect histopathological correlates of disease activity in FSHD. The molecular-pathological correlates to these changes warrants further investigation.