Mitogen-activated protein kinases-dependent induction of hepatocyte growth factor production in human dermal fibroblasts by the antibiotic polymyxin B.

Hepatocyte growth factor (HGF) stimulates migration and proliferation of keratinocytes and has been suggested to be involved in wound healing. The cationic antibiotic polymyxin B (PMB) is commonly used as a topical antibiotic for wound care. If PMB possesses an HGF-inducing activity, the antibiotic is potentially beneficial for wound healing in addition to minimizing chances of infection. In this study, we found that PMB markedly induced HGF production from various types of cells including human dermal fibroblasts. Its effect was stronger than the effects of epidermal growth factor and cholera toxin and was comparable to the effect of 8-bromo-cAMP. Among the polymyxin family and polymyxin derivatives, colistin was also effective, whereas colistin methanesulfonate had only a marginal effect and PMB nonapeptide was ineffective. The stimulatory effect of PMB was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) was observed from 0.25 h to 6h after the addition of PMB, while increase in phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected from 24h to 60 h after PMB addition. The MAPK/ERK kinase inhibitor PD98059, the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 all potently inhibited PMB-induced HGF production. Lastly, proliferation of human dermal fibroblasts was significantly stimulated by PMB. These results indicate that PMB-induced HGF production and proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the induction of HGF production.

Induction of hepatocyte growth factor production in human dermal fibroblasts and their proliferation by the extract of bitter melon pulp.

Hepatocyte growth factor (HGF) is useful as a potential therapeutic agent for hepatic and renal fibrosis and cardiovascular diseases through inducing proliferation of epithelial and endothelial cells. HGF inducers may also be useful as therapeutic agents for these diseases. However, there have been no reports on induction of HGF production by plant extracts or juices. An extract of bitter melon (Momordica charantia L.) pulp markedly induced HGF production. There was a time lag of 72 h before induction of HGF production after the extract addition. Its stimulatory effect was accompanied by upregulation of HGF gene expression. Increases in mitogen-activated protein kinases (MAPKs) were observed from 72 h after the extract addition. Inhibitors of MAPKs suppressed the extract-induced HGF production. The extract also stimulated cell proliferation. Both activities for induction of HGF production and cell proliferation were eluted together in a single peak with 14,000 Da on gel filtration. The results indicate that bitter melon pulp extract induced HGF production and cell proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the HGF induction. Our findings suggest potential usefulness of the extract for tissue regeneration and provide an insight into the molecular mechanism underlying the wound-healing property of bitter melon.

Induction of three vitellogenins by 17beta-estradiol with concurrent inhibition of the growth hormone-insulin-like growth factor 1 axis in a euryhaline teleost, the tilapia (Oreochromis mossambicus).

The objective of the present study was to utilize the male Mozambique tilapia (Oreochromis mossambicus) as a model for examining the molecular mechanisms that mediate the physiological transition between somatic and gonadal growth in female teleost fish, and in vertebrates in general. Partial cDNAs that encode multiple forms of vitellogenin (Vtg), which is the major precursor of yolk proteins, were cloned from estrogen-treated males and utilized to develop real-time quantitative RT-PCR assays, which were supplemented by an assay for Vtg immunoreactivity in the plasma. Alignment analyses of the amino acid sequences deduced from the vtg cDNAs revealed three distinct tilapia Vtgs, which were categorized as Aa-, Ab-, and C-type Vtgs. A single injection of male tilapias with 17beta-estradiol (E(2)) at 5 microg/g body weight significantly increased the plasma E(2) and hepatic levels of all three vtg transcripts within 1 day. Plasma E(2) levels declined after 3 days, whereas the plasma Vtg immunoreactivity and hepatic levels of the three vtg transcripts continued to increase. Hepatic expression of the estrogen receptor (esr) 1 gene, but not the esr2 gene, also increased markedly 1 day after E(2) injection and remained elevated for 5 days. While plasma growth hormone (Gh) levels were unaffected, hepatic expression of transcripts that encoded the Gh receptor and insulin-like growth factor 1 (Igf1) was suppressed by E(2), as were the plasma Igf1 levels. These results clearly suggest a distinct negative interplay between the growth and reproductive axes at the molecular level of key hepatic regulatory pathways involved in the control of energy utilization by gonadal and somatic growth processes.

Inhibition of hepatocyte growth factor production in human fibroblasts by ursodeoxycholic acid.

Hepatocyte growth factor (HGF) stimulates the proliferation of hepatocytes and biliary epithelial cells and protects hepatocytes from apoptosis induced by various stimuli. In view of HGF induction by interferons, substances used for the treatment of chronic hepatitis C, this study was conducted to determine whether ursodeoxycholic acid (UDCA), which is widely used for the treatment of cholestatic liver diseases, modulates HGF production. UDCA did not induce HGF production in human dermal fibroblasts, but it potently inhibited phorbol-12-myristate-13-acetate (PMA)- and cholera-toxin-induced HGF production without affecting cell viability. The inhibitory effects of UDCA were as potent as those of transforming growth factor-beta1 and dexamethasone. Up-regulations of HGF gene expression induced by PMA and cholera toxin were also inhibited by UDCA. Moreover, UDCA dose-dependently inhibited high constitutive HGF production by MRC-5 cells without decreasing cell viability. Deoxycholate, chenodeoxycholate, taurochenodeoxycholate and glycochenodeoxycholate also inhibited cholera-toxin-induced HGF production at non-cytotoxic doses. UDCA, however, had no apparent effect on PMA-induced phosphorylation of mitogen-activated protein kinase, which is crucial for HGF induction by PMA. These results indicate that non-cytotoxic doses of UDCA inhibited constitutive and induced HGF production and suggest that UDCA supplemented with HGF or HGF inducers could have a more potential therapeutic effect.

2-Furoyl-LIGRL-NH2, a potent agonist for proteinase-activated receptor-2, as a gastric mucosal cytoprotective agent in mice.

1. Proteinase-activated receptor-2 (PAR(2)), expressed in capsaicin-sensitive sensory neurons, plays a protective role in gastric mucosa. The present study evaluated gastric mucosal cytoprotective effect of 2-furoyl-LIGRL-NH(2), a novel highly potent PAR(2) agonist, in ddY mice and in wild-type and PAR(2)-knockout mice of C57BL/6 background. 2. Gastric mucosal injury was created by oral administration of HCl/ethanol solution in the mice. The native PAR(2)-activating peptide SLIGRL-NH(2), administered intraperitoneally (i.p.) at 0.3-1 micromol kg(-1) in combination with amastatin, an aminopeptidase inhibitor, but not alone, revealed gastric mucosal protection in ddY mice, which was abolished by ablation of capsaicin-sensitive sensory neurons. 3. I.p. administration of 2-furoyl-LIGRL-NH(2) at 0.1 micromol kg(-1), without combined treatment with amastatin, exhibited gastric mucosal cytoprotective activity in ddY mice, the potency being much greater than SLIGRL-NH(2) in combination with amastatin. This effect was also inhibited by capsaicin pretreatment. 4. Oral administration of 2-furoyl-LIGRL-NH(2) at 0.003-0.03 micromol kg(-1) also protected against gastric mucosal lesion in a capsaicin-reversible manner in ddY mice. 5. I.p. 2-furoyl-LIGRL-NH(2) at 0.1-0.3 micromol kg(-1) caused prompt salivation in anesthetized mice, whereas its oral administration at 0.003-1 micromol kg(-1) was incapable of eliciting salivation. 6. In wild-type, but not PAR(2)-knockout, mice of C57BL/6 background, i.p. administration of 2-furoyl-LIGRL-NH(2) caused gastric mucosal protection. 7. Thus, 2-furoyl-LIGRL-NH(2) is considered a potent and orally available gastric mucosal protective agent. Our data also substantiate a role for PAR(2) in gastric mucosal protection and the selective nature of 2-furoyl-LIGRL-NH(2).

A protective role of protease-activated receptor 1 in rat gastric mucosa.

BACKGROUND AND AIMS: On activation, protease-activated receptor (PAR)-2 modulates multiple gastric functions and exerts mucosal protection via activation of sensory neurons. The role of PAR-1, a thrombin receptor, in the stomach remains unknown. We thus examined if the PAR-1 agonist could protect against gastric mucosal injury in rats. METHODS: Gastric mucosal injury was created by oral administration of ethanol/HCl or absolute ethanol in conscious rats. Gastric mucosal blood flow and acid secretion were determined in anesthetized rats. Immunohistochemical analyses of PAR-1 and cyclooxygenase (COX)-1 were also performed in rat and human stomach. RESULTS: The PAR-1 agonist TFLLR-NH(2), administered intravenously in combination with amastatin, protected against the gastric mucosal injury induced by ethanol/HCl or absolute ethanol. The protective effect of TFLLR-NH(2) was abolished by indomethacin or a COX-1 inhibitor but not by ablation of sensory neurons with capsaicin. TFLLR-NH(2) produced an NO-independent increase in gastric mucosal blood flow that was partially inhibited by blockade of the endothelium-derived hyperpolarizing factor pathway. This inhibitory effect was promoted by indomethacin. TFLLR-NH(2) suppressed carbachol-evoked acid secretion in an indomethacin-reversible manner. Immunoreactive PAR-1 and COX-1 were expressed abundantly in rat gastric muscularis mucosae and smooth muscle, and the former protein was also detectable in blood vessels. Similar staining was observed in human gastric muscularis mucosae. CONCLUSIONS: The PAR-1 agonist, given systemically, protects against gastric mucosal injury via COX-1-dependent formation of prostanoids, modulating multiple gastric functions. Our data identify a novel protective role for PAR-1 in gastric mucosa, and the underlying mechanism is entirely different from that for PAR-2.

The PAR-1-activating peptide facilitates pepsinogen secretion in rats.

Protease-activated receptor-2 (PAR-2) is abundantly expressed in gastric mucosal chief cells, facilitating pepsinogen secretion. In the present study, we investigated whether PAR-1, a thrombin receptor, could modulate pepsinogen secretion in rats. The PAR-1-activating peptide TFLLR-NH(2) as well as the PAR-2-activating peptide SLIGRL-NH(2), administered i.v. repeatedly at 1-h intervals, significantly increased gastric pepsinogen secretion over 2-4 h (after two to four doses). In contrast, the control peptide FTLLR-NH(2), given in the same manner, had no such effect. Thus, PAR-1, like PAR-2, might function to facilitate pepsinogen secretion, suggesting a novel role of the thrombin-PAR-1-pathway in the stomach.

Vitellogenin-derived yolk proteins of white perch, Morone americana: purification, characterization, and vitellogenin-receptor binding.

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.

Vitellogenin-derived yolk proteins in a hybrid sturgeon, bester (Huso huso x Acipencer ruthenus): identification, characterization and course of proteolysis during embryogenesis.

Vitellogenin (Vg) and its corresponding yolk protein (YP) products, YP1, YP2 and YP3, were isolated from serum of estrogen-treated hybrid sturgeon (bester; Huso huso X Acipencer ruthenus) and eggs from untreated fish, respectively. Vitellogenin had an apparent molecular mass of 580 kDa and appeared as two major bands corresponding to 180 kDa and 120 kDa after SDS-PAGE. Apparent molecular weights of YP1, YP2 and YP3 were 370 kDa, 88 kDa and 19 kDa, respectively. After SDS-PAGE, YP1 appeared as a main band of 110 kDa, while YP2 was resolved as a single band of 94 kDa and 29 kDa band under non-reducing and reducing conditions, respectively. Yolk protein 3 appeared as a diffuse band corresponding to 16 kDa and two faint bands below 14.4 kDa after SDS-PAGE. However, the 16 kDa band alone was observed after dephosphorylation with alkaline phosphatase. The course of cleavage of yolk proteins in bester embryos and alevins was observed by SDS-PAGE and Western blotting from fertilization onward. After hatching, the main 110 kDa band of YP1 was degraded into smaller peptides during development, while YP2 hardly showed any such structural changes. The amino acid compositions of purified yolk proteins indicated that YP1, YP2 and YP3 were bester lipovitellin, beta-component, and phosvitin, respectively.