Kouji Okamoto

Modulation by elastin peptide VGVAPG of cell proliferation and elastin expression in human skin fibroblasts

Elastin is a major component of dermal connective tissue and confers elasticity to the tissues [1]. Elastin has unique repeating sequences in the hydrophobic region: the pentapeptide VPGVG and the hexapeptide XPGVGV (X = 1 or V). The pentapeptide VPGVG is the only repeating sequence present in the elastin molecules of all animal species so far analyzed including human, bovine, porcine and chicken elastin [2–6]. VGVAPG is a hexapeptide repeated many times in human, bovine and porcine elastin molecules, but not present in the chicken elastin molecule [2, 7]. The hydrophobic repeating peptides of elastin VGVAPG are able to generate directed cell movements of human monocytes, bovine ligamentum nuchae fibroblasts [8], aortic endothelial cells [9] and murine lung carcinoma cells [10]. The interaction of the elastin peptides with these cells is thought to be mediated by a high-affinity cell receptor for VGVAPG sequences, which is related to membrane-bound protein kinase C (PKC) activity [10, 11]. We have previously reported that synthetic elastin peptide VPGVG stimulates the proliferation of chick vascular smooth muscle cells and downregulates elastin expression [12]. In this study, we evaluated the effects of two synthetic elastin peptides VPGVG and VGVAPG, both of which are present in human elastin molecules, on the biosynthetic phenotype of extracellular matrix proteins and on the proliferation of human dermal fibroblasts. The following chemicals were purchased from Seikagaku Corporation, Tokyo: the inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) [13]; the inhibitor for cAMPand cGMP-dependent protein kinase, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004) [14]; the calcium-calmodulin binding inhibitor, N-(6-amino-hexyl)-5-chloro1naphthalenesulfonamide (W-7) [15], and the inhibitor for cAMP-dependent protein kinase, (N-[2-(P-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89) [16]. Heparin and 2-O-tetradecanoylphorbol 13-acetate (TPA) were purchased from Sigma. The peptides, VPGVG and VGVAPG were synthesized by a solid-phase method. Polymers of VPGVG and VGVAPG [(VPGVG)n, and (VGVAPG)n, respectively] were synthesized as previously deShingo Tajima · Hiroshi Wachi · Yuko Uemura · Kouji Okamoto

Stimulation of cell proliferation and autoregulation of elastin expression by elastin peptide VPGVG in cultured chick vascular smooth muscle cells

Synthetic elastin peptides, VPGVG or its polymer (VPGVG) n , enhanced the proliferation of smooth muscle cells 1.5‐fold during 48 h treatment at the concetrations over 10−6 M or 1.0 μg/ml, respectively. Monomeric and polymeric VPGVG sequences reduced elastin synthesis and its mRNA level to one‐third and one‐half of control respectively under the conditions in which the proliferation of cells were enhanced, but did not change collagen synthesis as measured by bacterial collagenase digestion. The elastin‐specific autoregulation by elastin fragments may reflect the feedback regulation of elastin expression which may play an essential role in elastin metabolism under the normal and diseased conditions.

Elastin‐derived peptide induces monocyte chemotaxis by increasing intracellular cyclic GMP level and activating cyclic GMP dependent protein kinase

An elastin‐derived peptide with an average molecular mass of 25 kDa was shown to induce monocyte chemotaxis at the optimal concentration of 10‐1 μg/ml. Homologous deactivation test showed that monocytes exposed to the elastin‐derived peptide at 10‐1 μg/ml lost their chemotactic responsiveness when reexposed to the same stimulus. In conjunction with chemotactic response to the elastin‐derived peptide, intracellular guanosine 3′, 5′‐monophosphate (cGMP) levels were enhanced but intracellular adenosine 3′, 5′‐monophosphate (cAMP) levels were not. The monocyte migration induced by the elastin‐derived peptide was inhibited by cGMP dependent protein kinase (PKG) inhibitor, but not by cAMP dependent protein kinase inhibitor and protein kinase C inhibitor. These results suggest that the elastin‐derived peptide induces monocyte chemotaxis by increasing the level of cGMP, followed by activating PKG.

Expression of 36-kDa microfibril-associated glycoprotein (MAGP-36) in human keratinocytes and its localization in skin

Microfibril-associated glycoprotein-36 (MAGP-36) is a recently isolated elastin-binding protein and considered to be a member of microfibril-associated glycoproteins (MAGPs). We studied the expression of MAGP-36 in cultured normal human keratinocytes and its localization in the skin. MAGP-36 was found to be expressed in cultured human keratinocytes by Western blot and RT-PCR assays. The levels of MAGP-36 (polypeptide and mRNA) and the number of MAGP-36-producing keratinocytes were greatly increased during Ca(2+)-induced differentiation of keratinocytes. Immunohistochemical studies demonstrated that MAGP-36 colocalized with elastic fibers and formed candelabra like-fibers in the superficial dermis of normal skin. In the elderly skin of sun-exposed region, immunoreactivity of MAGP-36 in the superficial dermis disappeared. In the lesional skin of pseudoxanthoma elasticum which is an elastin-related disorder, immunoreactivity of MAGP-36 was found in the accumulation of disintegrated elastic fibers. The results show that MAGP-36 is a component of elastic fibers in the dermis and co-operates with elastin in normal and diseased conditions.

Inhibitory Effect of Gramicidin S on the Growth of Murine Tumor Cells in vitro and in vivo

The Effect of gramicidin S (GS), a polypeptide antibiotic, on the growth of murine tumor cells such as allotransplantable sarcoma 180 (S180) and Ehrlich ascites carcinoma (EAC) and Meth A fibrosarcoma (Meth A) was studied. GS inhibited the proliferation of EAC cells in culture and its effect on cell viability was dependent on the concentration of GS. On exposure to GS at concentrations ranging from 1 to 100 micrograms/ml for 10 min, EAC cells lost their transplantability in ddY mice depending on the concentration of GS. In particular, the transplantability of EAC cells was completely missing on exposure to GS at a concentration of 100 micrograms/ml. In the in vivo experiments, a daily intraperitoneal injection of GS exhibited a high inhibitory effect not only on the growth of subcutaneously implanted S180 in ICR mice but on the growth of subcutaneously implanted syngeneic Meth A in BALB/c mice. In the studies using radioactively labeled DNA, RNA and protein precursors, GS at high concentrations inhibited the incorporation of all the precursors into EAC cells.