The involvement of transforming growth factor-beta isoforms in the induction of the regressing phase (catagen) of human hair follicles were examined in vivo. In the growing phase (anagen), transforming growth factor-beta1 was detected at the hair cuticle and connective tissue sheath. Transforming growth factor-beta2 was restricted to the outermost cell layer of the outer root sheath. Transforming growth factor-beta3 was observed in the precortical hair matrix of anagen hair follicles. During the anagen-catagen transition phase, strong transforming growth factor-beta2 immunoreactivity appeared in the lower bulb matrix cells adjacent to the dermal papilla. In addition, transforming growth factor-beta2 and transforming growth factor-beta type II receptor were colocalized in the regressing epithelial strands, where terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive apoptotic cells were also found. Transforming growth factor-beta1 and transforming growth factor-beta3 were mostly negative in the strand. Using an organ culture system, we investigated whether transforming growth factor-beta2 and its antagonists affected the transition process. Elongation of hair was significantly suppressed by transforming growth factor-beta2. Next, a neutralizing antibody and fetuin, a potent transforming growth factor-beta antagonist was tested. In the presence of the antibody as well as fetuin, hair follicles were markedly elongated in a concentration-dependent manner. These results strongly suggest that transforming growth factor-beta2 plays an essential part in the induction of the catagen phase of the human hair cycle.

TGF-beta plays important roles in the induction of catagen during the hair cycle. We examined whether TGF-beta2 could activate a caspase in human hair follicles. Using active caspase-9 and -3 specific antibodies, we found that TGF-beta2 activated these caspases in two regions, the lower part of the hair bulb and the outer layer of the outer root sheath. In addition, we searched for a plant extract that can effectively suppress TGF-beta action. We found that an extract of Hydrangea macrophylla reduced synthesis of a TGDbeta-inducible protein. We confirmed that the extract has a potential to promote hair elongation in the organ culture system. Furthermore, it delayed in vivo progression of catagen in a mouse model. Our results suggest that the induction of catagen by TGF-beta is mediated via activation of caspases and that a suppressor of TGF-beta could be effective in preventing male pattern baldness.

Restricted expression of caspase-14 in differentiating keratinocytes suggests the involvement of caspase-14 in terminal differentiation. We purified active caspase-14 from human cornified cells with sequential chromatographic procedures. Specific activity increased 764-fold with a yield of 9.1%. Purified caspase-14 revealed the highest activity on WEHD-methylcoumaryl-amide (MCA), although YVAD-MCA, another caspase-1 substrate, was poorly hydrolyzed. The purified protein was a heterodimer with 17 and 11 kDa subunits. N-terminal and C-terminal analyses demonstrated that the large subunit consisted of Ser(6)-Asp(146) and N-terminal of small subunit was identified as Lys(153). We successfully developed an antiserum (anti-h14D146) directed against the Asp(146) cleavage site, which reacted only with active caspase-14 but not with procaspase-14. Furthermore we confirmed that anti-h14D146 did not show any reactivity to the active forms of other caspases. Immunohistochemical analysis demonstrated that anti-h14D146 staining was mostly restricted to the cornified layer and co-localized with some of the TUNEL positive-granular cells in the normal human epidermis. UV radiation study demonstrated that caspase-3 was activated and co-localized with TUNEL-positive cells in the middle layer of human epidermis. In contrast, we could not detect caspase-14 activation in response to UV. Our study revealed tightly regulated action of caspase-14, in which only the terminal differentiation of keratinocytes controls its activation process.

We purified a novel serine protease with a molecular mass of 26 kDa from Sarcophaga pupae. This protease appeared almost exclusively in the yellow body, an organ that develops temporarily in the pupae of dipteran insects and expands to form the adult midgut by engulfing the larval midgut. cDNA analysis revealed that this protease consists of 239 amino acid residues and has significant structural similarity with bovine trypsin (about 40% sequence identity). The 26-kDa protease gene was transiently activated in 1-day-old pupae. The protease was found to cross-react immunologically with antibody against sarcotoxin IA, an antibacterial protein produced by this insect. It is suggested that this protease participates in the decomposition of the larval midgut in the yellow body during metamorphosis.

We have reported a novel serine protease produced by Sarcophaga peregrina (Nakajima et al., J. Biol. Chem. 272 (1997) 23805^23810). This 26-kDa protease showed antibacterial activity against several bacteria. This activity was an intrinsic characteristic of the enzyme protein and not directly related to its protease activity, because treating the 26-kDa protease with diisopropyl fluorophosphate had no appreciable effect on its antibacterial activity. Unlike bovine trypsin, the 26-kDa protease interacted with acidic phospholipids, suggesting that its antibacterial activity is attributable to interaction with bacterial membranes.z 1998 Federation of European Biochemical Societies.