Chie Morimoto

Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing

Members of the epidermal growth factor (EGF) family are the most important growth factors involved in epithelialization during cutaneous wound healing. Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is thought to play an important role in skin wound healing. To investigate the in vivo function of HB-EGF in skin wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter. Studies of wound healing revealed that wound closure was markedly impaired in keratinocyte-specific HB-EGF-deficient mice. HB-EGF mRNA was upregulated at the migrating epidermal edge, although cell growth was not altered. Of the members of the EGF family, HB-EGF mRNA expression was induced the most rapidly and dramatically as a result of scraping in vitro. Combined, these findings clearly demonstrate, for the first time, that HB-EGF is the predominant growth factor involved in epithelialization in skin wound healing in vivo and that it functions by accelerating keratinocyte migration, rather than proliferation.

Membrane-anchored growth factors, the epidermal growth factor family: beyond receptor ligands.

The epidermal growth factor (EGF) family and the EGF receptor (EGFR, ErbB) tyrosine kinase family have been spearheading the studies of signal transduction events that determine cell fate and behavior in vitro and in vivo. The EGFR family and their signaling pathways are giving us tremendous advantages in developing fascinating molecular target strategies for cancer therapy. Currently, two important types of EGFR inhibitors are in clinical use: neutralizing antibodies of EGFR or ErbB2, and synthetic small compounds of tyrosine kinase inhibitors designed for receptors. On the other hand, basic research of the EGF family ligands presents new challenges as membrane‐anchored growth factors. All members of the EGF family have important roles in development and diseases and are shed from the plasma membrane by metalloproteases. The ectodomain shedding of the ligands has emerged as a critical component in the functional transactivation of EGFRs in interreceptor cross‐talk in response to various shedding stimulants such as G‐protein coupled receptor agonists, growth factors, cytokines, and various physicochemical stresses. Among the EGFR‐ligands, heparin‐binding EGF‐like growth factor (HB‐EGF) is a prominent ligand in our understanding of the pathophysiological roles of ectodomain shedding in cancer, wound healing, cardiac diseases, etc. Here we focus on ectodomain shedding of the EGF family ligands, especially HB‐EGF by disintegrin and metalloproteases, which are not only key events of receptor cross talk, but also novel intercellular signaling by their carboxy‐terminal fragments to regulate gene expression directly. (Cancer Sci 2008; 99: 214–220)

Biomodulator-mediated susceptibility of endogenous lipid droplets from rat adipocytes to hormone-sensitive lipase.

The amount of fatty acid release by a fat cell homogenate without pretreatment with epinephrine was found to be slightly more than that released from fat cells by epinephrine, suggesting that fat cells contain high lipolytic activity even in the absence of lipolytic agents. Fat cells contain high hormone-sensitive lipase activity (1383 mumole free fatty acids/g/hr) in the absence of epinephrine, and addition of epinephrine to the cells did not increase the activity, significantly. Like epinephrine, DBcAMP and/or theophylline also elicited marked release of glycerol from fat cells without activating the hormone-sensitive lipase activity. However, although fat cells contain a large amount of hormone-sensitive lipase, lipolysis was negligible in the absence of these lipolytic agents. These results suggest that lipolytic agents such as epinephrine, DBcAMP, and theophylline induce lipolysis in fat cells through some mechanism other than activation of hormone-sensitive lipase and that in the absence of lipolytic agents, some system in fat cells inhibits lipolysis of endogenous lipid droplets by hormone-sensitive lipase. The lipid droplets in fat cells consist mainly of triglyceride with phospholipids, cholesterol, carbohydrate, and protein as minor constituents. The phospholipid fraction was found to consist of 75% phosphatidylcholine and 25% phosphatidylethanolamine. Of the minor constituents of endogenous lipid droplets, only phosphatidylcholine strongly inhibited hormone-sensitive lipase activity in a [3H]triolein emulsion. These results suggest that phosphatidylcholine in endogenous lipid droplets may be responsible for inhibition of hormone-sensitive lipase. Then, a cell-free system was established in which epinephrine, DBcAMP, and theophylline stimulated lipolysis of endogenous lipid droplets from fat cells by lipase solution. In this system, these lipolytic agents did not induce lipolysis in the absence of added lipase. Lipolysis in the mixture of the endogenous lipid droplets and lipase solution was accelerated by phospholipase C with concomitant loss of epinephrine-induced lipolysis. After pretreatment of the endogenous lipid droplets with phospholipase C, these lipolytic agents no longer induced lipolysis. Pretreatment of the endogenous lipid droplets with phospholipase C reduced their phospholipid content with the formation of phosphorylcholine, but did not affect their triglyceride and cholesterol contents. Treatment of the endogenous lipid droplets with phospholipase D did not affect lipolysis in the cell-free system. These results suggest that phosphatidylcholine in the endogenous lipid droplets may inhibit their lipolysis by hormone-sensitive lipase in fat cells and also be involved in the mechanisms of the stimulatory effects of epinephrine, DBcAMP, and theophylline on lipolysis.

Propranolol-sensitive and phenoxybenzamine-insensitive binding of norepinephrine to endogenous lipid droplets from rat adipocytes.

Norepinephrine, epinephrine, and isoproterenol at concentrations of 5.5 x 10(-8) M were found to elicit lipolysis in a cell-free system containing lipid droplets from fat cells and lipase solution. In the cell-free system, the beta-blockers propranolol and dichloroisoproterenol at concentrations of 1 microM inhibited lipolysis induced by norepinephrine, whereas similar concentrations of the alpha-blockers phenoxybenzamine and yohimbine did not inhibit lipolysis. The binding of norepinephrine to endogenous lipid droplets was inhibited by propranolol, but not by phenoxybenzamine. We concluded that the propranolol-sensitive, phenoxybenzamine-insensitive binding of norepinephrine to endogenous lipid droplets is involved in lipolysis in fat cells. Treatment of endogenous lipid droplets with phospholipase C, but not phospholipase D, trypsin, chymotrypsin, or neuraminidase, inhibited the propranolol-sensitive binding of norepinephrine to the droplets. These results suggest that the phosphate group of phospholipid in endogenous lipid droplets may be the site of propranolol-sensitive binding of norepinephrine. The physiological significance of the propranolol-sensitive binding is discussed.

ESR studies of coenzyme Q1, chromanoxyl and chromenoxyl radicals

ESR measurements were performed for the coenzyme Q1, chromanoxyl and chromenoxyl radicals obtained by oxidizing the phenol precursors with PbO2 in toluene, and the proton hyperfine splittings were correctly determined.

ESR studies of vitamin K1 chromanoxyl and chromenoxyl radicals

Abstract The ESR spectra of vitamin K 1 chromanoxyl and chromenoxyl radicals were observed by oxidizing the phenol precursors with PbO 2 in toluene, and the proton hyperfine splittings and g iso -values were correctly determined.

Mechanism of norepinephrine-induced lipolysis in isolated adipocytes: evidence for its lipolytic action inside the cells

Norepinephrine elicited remarkable lipolysis in rat fat cells, but did not stimulate lipolysis in a fat cell homogenate. We prepared endogenous lipid droplets from fat cells and established a norepinephrine-sensitive cell-free system consisting of the lipid droplets and hormone-sensitive lipase (HSL). In the cell-free system, hydrolysis of the lipid droplets was markedly increased by addition of norepinephrine but homogenization of the droplets caused loss of norepinephrine responsiveness. These results suggest that the site of the lipolytic action of norepinephrine may be the lipid droplets. When 3T3-L1 fat cell monolayers were exposed to 3H-norepinephrine for 2 or 4 min, many silver grains, representing the localization of labeled norepinephrine, were observed in autoradiograms of cell sections. The grains were found in the cytoplasm and along the margin of the lipid droplets. These results also suggest that norepinephrine may be incorporated into fat cells and exhibit its lipolytic action inside the cells.