(Bulb) Hair bulb; (epi) epidermis; (mat) matrix; (ORS) outer root sheath. at approximately P10 in the hair matrix and ORS, and peaks in late anagen (Hebert et al. 1994). By in situ hybridization, we find that FGF5 is usually up-regulated in a subset of hair follicles at P7 in mRNA is normally expressed during anagen and is down-regulated at catagen entry (Cho et al. 2003). In situ hybridization shows that PTHRP is usually down-regulated at P12 in shows the percentage of apoptotic cells based on TUNEL and Hoechst staining, while shows a caspase activity assay (RFU, relative fluorescence models). Both and show that 0.01. (cDNA (cells. 0.001. To assess whether K17 acts cell-autonomously in this setting, 0.001), as expected (Fig. ?(Fig.2E).2E). Together, these findings show that loss of K17 cell-autonomously increases the susceptibility of cultured skin keratinocytes to TNF-mediated apoptosis, suggesting that such may be the case as well in vivo. K17 interacts with TRADD, a death adaptor, in mouse skin keratinocytes That K17, a cytoskeletal protein, influences the responsiveness of keratinocytes to TNF is a priori surprising. Aceglutamide The previously documented interaction between K18 and TRADD (Inada et al. 2001), a death adaptor protein essential for signal relay downstream from TNF receptor 1 (TNFR1) (Micheau and Tschopp 2003), provides a possible mechanism. Inada et al. (2001) found that the K18CTRADD interaction affords a protection against TNF-induced apoptosis, possibly via the sequestration of TRADD away from TNFR1. TRADD also binds K14 in a human keratinocyte cell line (Yoneda et al. 2004). Mapping studies showed Aceglutamide that TRADD binds the 1A subdomain in K18 (Inada Aceglutamide et al. 2001) and K14 (Yoneda et al. 2004), a region that is highly conserved in other type I keratins. The physiological significance of these interactions in vivo is unknown. Coimmunoprecipitation assays using two distinct antibodies revealed that, as expected, K17, K16, and K14 are present in endogenous TRADD immunoprecipitates obtained from primary cultures of mouse skin keratinocytes (Fig. 3A, A). In mouse keratinocytes transfected with GFP fusion constructs, both full-length TRADD (residues 1C312) (Fig. 3BCB) and its C-terminal moiety (residues 105C312) (Fig. 3CCC) partially colocalize with K17 in the cytoplasm. Similar results were obtained in preparations double-stained for K14 and TRADD (data not shown). In contrast, the N terminus of TRADD (residues 1C105) shows a diffuse distribution in the cytoplasm and nucleus (data not shown). Partial colocalization between TRADD, a signaling adaptor present at low levels, and the abundant K17 does not preclude a physiologically relevant interaction. The finding that TRADD potentially interacts with K14 and K16 in vivo is significant, as K16 has been implicated in the reversibility and strain-dependence of the hair phenotype in The migration of 37- and 49-kDa markers is shown at (corner. Arrowheads point Aceglutamide to instances of colocalization. Bars, 30 m. TNF signaling contributes to hair cycle regulation in vivo There is as yet no definitive evidence implicating the TNF/TNFR1 signaling pathway in mature hair follicles of postnatal mouse skin. Several conditions need to be met to support the possibility that TNF/TNFR1 play a role in hair cycling, and specifically, in the defect exhibited by (Fig. 4B, B) and (Fig. 4C, C) mRNAs occur in the hair matrix and ORS compartments of anagen-stage wild-type follicles, in a pattern that overlaps significantly with that of Keratin 7 antibody K17 (Fig. 4A, A). Open in a separate window Figure 4. Characterization of TNF signaling in wild-type mouse skin. ((((and are shown at higher magnification in and respectively. Asterisks denote melanin pigment. Arrows point to hybridization signals. (Bulb) Hair bulb; (epi).