Immunohistochemical Characterization of Facioscapulohumeral Muscular Dystrophy Muscle Biopsies.

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.

PGE(2) is a UVR-inducible autocrine factor for human melanocytes that stimulates tyrosinase activation.

Prostaglandins activate signalling pathways involved in growth, differentiation and apoptosis. Prostaglandin E(2) (PGE(2)) is released by keratinocytes following ultraviolet irradiation (UVR) and stimulates the formation of dendrites in melanocytes. We show that multiple irradiations of human melanocytes with UVR-activated cPLA(2), the rate-limiting enzyme in eicosanoid synthesis and stimulated PGE(2) secretion. PGE(2) increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE(2) binds to four distinct G-protein coupled receptors (EP(1-4)). We show that PGE(2) stimulates EP(4) receptor signalling in melanocytes, resulting in cAMP production. Conversely, PGE(2) also stimulated the EP(3) receptor in melanocytes, resulting in lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE(2) on cAMP in melanocytes. The data are the first to identify PGE(2) as an UVR-inducible autocrine factor for melanocytes. These data also show that PGE(2) activates EP(3) and EP(4) receptor signalling, resulting in opposing effects on cAMP production, a critical signalling pathway that regulates proliferation and melanogenesis in melanocytes.

Plexin B1 suppresses c-Met in melanoma: a role for plexin B1 as a tumor-suppressor protein through regulation of c-Met.

Melanoma arises through complex genetic and epigenetic changes, resulting in uncontrolled proliferation, invasion, and metastatic disease. Semaphorins regulate axon guidance through interaction with their receptors, plexins and neuropilins. Plexin B1, the semaphorin 4D receptor, activates oncogenic receptors c-Met and ErbB-2 in several cell types, suggesting it promotes tumor growth through stimulation of these receptors. A study by Argast et al. has shown that plexin B1 is a tumor-suppressor protein for melanoma metastasis in a mouse model. In this report, we show that plexin B1 is lost in metastatic and deeply invasive melanoma in patient samples in vivo. Unexpectedly, introduction of plexin B1 into human melanoma cell lines suppressed, rather than activated, the oncogenic receptor, c-Met, by its ligand hepatocyte growth factor (HGF). Plexin B1 also activated Akt in melanoma. Plexin B1 significantly abrogated cell migration in response to HGF but rendered cells resistant to apoptosis by cisplatin. Plexin B1 is predicted to function as a classic tumor-suppressor protein in melanoma, in part through suppression of c-Met signaling and c-Met-dependent migration. However, because plexin B1 activates Akt, a multifunctional protein involved in tumor progression in several cancers, plexin B1 may function as a tumor promoter in melanomas not driven by c-Met activation.

Plexin C1, a receptor for semaphorin 7a, inactivates cofilin and is a potential tumor suppressor for melanoma progression.

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.

Semaphorin 7a promotes spreading and dendricity in human melanocytes through beta1-integrins.

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.

Prostaglandin E2 regulates melanocyte dendrite formation through activation of PKCzeta.

Prostaglandins are lipid signaling intermediates released by keratinocytes in response to ultraviolet irradiation (UVR) in the skin. The main prostaglandin released following UVR is PGE(2), a ligand for 4 related G-protein-coupled receptors (EP(1), EP(2), EP(3) and EP(4)). Our previous work established that PGE(2) stimulates melanocyte dendrite formation through activation of the EP(1) and EP(3) receptors. The purpose of the present report is to define the signaling intermediates involved in EP(1)- and EP(3)-dependent dendrite formation in human melanocytes. We recently showed that activation of the atypical PKCzeta isoform stimulates melanocyte dendricity in response to treatment with lysophosphatidylcholine. We therefore examined the potential contribution of PKCzeta activation on EP(1)- and EP(3)-dependent dendrite formation in melanocytes. Stimulation of the EP(1) and EP(3) receptors by selective agonists activated PKCzeta, and inhibition of PKCzeta activation abrogated EP(1)- and EP(3)-receptor-mediated melanocyte dendricity. Because of the importance of Rho-GTP binding proteins in the regulation of melanocyte dendricity, we also examined the effect of EP(1) and EP(3) receptor activation on Rac and Rho activity. Neither Rac nor Rho was activated upon treatment with EP(1,3)-receptor agonists. We show that melanocytes express only the EP(3A1) isoform, but not the EP(3B) receptor isoform, previously associated with Rho activation, consistent with a lack of Rho stimulation by EP(3) agonists. Our data suggest that PKCzeta activation plays a predominant role in regulation of PGE(2)-dependent melanocyte dendricity.