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Dermal papilla (DP) at the hair follicle base is important for hair growth. Recent studies demonstrated that mouse vibrissa DP cells can be cultured in the presence of fibroblast growth factor-2 (FGF-2), but lose expression of versican and their follicle-inducing activity during the culture, and that activation of the Wnt signal, which is inhibited by glycogen synthase kinase-3 (GSK-3), in the DP cells promotes hair growth activity. We therefore investigated the influence of a GSK-3 inhibitor, (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), on the growth of human DP cells and mouse vibrissa follicles in culture. We first demonstrated that, similarly to mouse DP cells, human DP cells were able to be cultured up to 15 passages in the presence of FGF-2, and lost the expression of alkaline phosphatase (ALP). When human DP cells later than ten passages were treated with BIO, the expression of ALP as well as insulin-like growth factor-1 (IGF-1), another DP marker, was significantly elevated. Nuclear and perinuclear translocation of beta-catenin was also observed. We then cultured mouse vibrissa follicles. In the presence of BIO, the follicles could be maintained for at least 3 days without detectable regression of the hair bulbs. The morphology and ALP expression were well preserved. BIO successfully retrieved the expression of DP marker molecules, such as ALP and IGF-1 in cultured human DP cells, and maintained mouse hair bulbs. Thus, treatment with BIO may be useful to prepare DP cells with hair follicle-inducing activity.

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Mucin type O-glycosylation begins with the transfer of GalNAc to serine and threonine residues on proteins by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminlytransferases. These enzymes all contain a lectin-like (QXW) 3 repeat sequence at the C terminus that consists of three tandem repeats (␣, ␤, and ␥). The putative lectin domain of one of the most ubiquitous isozymes, GalNAc-T1, is reportedly not functional. In this report, we have reevaluated the role of the GalNAc-T1 lectin domain. Deletion of the lectin domain resulted in a complete loss of enzymatic activity. We also found that GalNAc-T1 has two activities distinguished by their sensitivities to inhibition with free GalNAc; one activity is sensitive, and the other is resistant. In our experiments, the former activity is represented by the O-glycosylation of apomucin, an acceptor that contains multiple glycosylation sites, and the latter is represented by synthetic peptides that contain a single glycosylation site. Site-directed mutagenesis of the lectin domain selectively reduced the former activity and identified Asp 444 in the ␣ repeat as the most important site for GalNAc recognition. A further reduction of the GalNAc-inhibitable activity was observed when both Asp 444 and the corresponding aspartate residues in the ␤ and the ␥ repeats were mutated. This suggests a cooperative involvement of each repeat unit in the glycosylation of polypeptides with multiple acceptor sites.O-Glycosidically linked oligosaccharides, called mucin-type oligosaccharides, are linked to polypeptides through an ␣-linkage (GalNAc␣1 3 Ser (or Thr)) and are known to occur on mucins as well as on other secretory and membrane glycoproteins (1). The initial step in the biosynthesis of these structures is catalyzed by a group of enzymes known as the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNActransferases).