The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C 2 -ceramide inhibited insulin-stimulated glucose transport by ϳ50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C 2 -ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C 2 -ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C 2 -ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C 2 -ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide's inhibition of Akt. These studies demonstrate ceramide's capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis.Insulin stimulates glucose uptake into muscle and adipose tissues by effecting the redistribution of the insulin-responsive glucose transporter GLUT4 from intracellular stores to the plasma membrane. Subsequently, insulin activates numerous metabolic pathways which promote the storage of the incoming glucose as glycogen or fat. Insulin transmits its signals through a cell surface tyrosine kinase receptor which stimulates multiple intracellular signaling events (reviewed in reference 41). Activated insulin receptors phosphorylate adapter proteins, such as members of the insulin receptor substrate (IRS) family, which recruit and activate downstream effector molecules. One of these proteins, phosphatidylinositol 3-kinase (PI 3-kinase), is requisite for insulin's acute regulation of glucose metabolism. Treatment with either of the PI 3-kinase inhibitors wortmannin or LY294002 blocks insulin's effects on glucose metabolism (6, 7, 35, 49), while expression of constitutively active forms of PI 3-kinase stimulates them (14,26,33). In single-cell assays, microinjection of dominant negative forms of PI 3-kinase (19, 31) or inhibitory PI 3-kinase antibodies (20) blocks G...

To determine whether endogenous synthesis of new extracellular matrix may contribute to the degree and duration of clinical benefits derived from crosslinked hyaluronic acid dermal filler injections. Design:In vivo biochemical analyses after filler injections.Setting: Academic referral center.Participants: Eleven healthy volunteers (mean age, 74 years) with photodamaged forearm skin.Interventions: Filler and vehicle (isotonic sodium chloride) injected into forearm skin and skin biopsy specimens taken 4 and 13 weeks later.Main Outcome Measures: De novo synthesis of collagen, the major structural protein of dermal extracellular matrix, was assessed using immunohistochemical analysis, quantitative polymerase chain reaction, and electron microscopy.Results: Compared with controls, immunostaining in skin receiving cross-linked hyaluronic acid injections re-vealed increased collagen deposition around the filler. Staining for prolyl-4-hydroxylase and the C-terminal and Nterminal epitopes of type I procollagen was enhanced at 4 and 13 weeks after treatment (PϽ.05). Gene expression for types I and III procollagen as well as several profibrotic growth factors was also up-regulated at 4 and 13 weeks compared with controls (PϽ.05). Fibroblasts in filler-injected skin demonstrated a mechanically stretched appearance and a biosynthetic phenotype. In vitro, fibroblasts did not bind the filler, suggesting that cross-linked hyaluronic acid is not directly stimulatory.

Androgenetic alopecia (AGA), also known as common baldness, is characterized by a marked decrease in hair follicle size, which could be related to the loss of hair follicle stem or progenitor cells. To test this hypothesis, we analyzed bald and non-bald scalp from AGA individuals for the presence of hair follicle stem and progenitor cells. Cells expressing cytokeratin15 (KRT15), CD200, CD34, and integrin, α6 (ITGA6) were quantitated via flow cytometry. High levels of KRT15 expression correlated with stem cell properties of small cell size and quiescence. These KRT15(hi) stem cells were maintained in bald scalp samples. However, CD200(hi)ITGA6(hi) and CD34(hi) cell populations--which both possessed a progenitor phenotype, in that they localized closely to the stem cell-rich bulge area but were larger and more proliferative than the KRT15(hi) stem cells--were markedly diminished. In functional assays, analogous CD200(hi)Itga6(hi) cells from murine hair follicles were multipotent and generated new hair follicles in skin reconstitution assays. These findings support the notion that a defect in conversion of hair follicle stem cells to progenitor cells plays a role in the pathogenesis of AGA.

Testosterone is necessary for the development of male pattern baldness, known as androgenetic alopecia (AGA); yet, the mechanisms for decreased hair growth in this disorder are unclear. We show that prostaglandin D2 synthase (PTGDS) is elevated at the mRNA and protein levels in bald scalp compared to haired scalp of men with AGA. The product of PTGDS enzyme activity, prostaglandin D2 (PGD2), is similarly elevated in bald scalp. During normal follicle cycling in mice, Ptgds and PGD2 levels increase immediately preceding the regression phase, suggesting an inhibitory effect on hair growth. We show that PGD2 inhibits hair growth in explanted human hair follicles and when applied topically to mice. Hair growth inhibition requires the PGD2 receptor G protein (heterotrimeric guanine nucleotide)–coupled receptor 44 (GPR44), but not the PGD2 receptor 1 (PTGDR). Furthermore, we find that a transgenic mouse, K14-Ptgs2, which targets prostaglandin-endoperoxide synthase 2 expression to the skin, demonstrates elevated levels of PGD2 in the skin and develops alopecia, follicular miniaturization, and sebaceous gland hyperplasia, which are all hallmarks of human AGA. These results define PGD2 as an inhibitor of hair growth in AGA and suggest the PGD2-GPR44 pathway as a potential target for treatment.

Regeneration of skin and hair follicles after wounding - a process known as wound-induced hair neogenesis (WIHN) - is a rare example of adult organogenesis in mammals. As such, WIHN provides a unique model system for deciphering mechanisms underlying mammalian regeneration. Here, we show that dsRNA, which is released from damaged skin, activates Toll-Like Receptor 3 (TLR3) and its downstream effectors IL6 and STAT3 to promote hair follicle regeneration. Conversely, TLR3-deficient animals fail to initiate WIHN. TLR3 activation promotes expression of hair follicle stem cell markers and induces elements of the core hair morphogenetic program, including EDAR and the Wnt and Shh pathways. Our results therefore show that dsRNA and TLR3 link the earliest events of mammalian skin wounding to regeneration and suggest potential therapeutic approaches for promoting hair neogenesis.