Induction of Mucous Metaplasia in Chick Embryonic Skin by Retinol-Pretreated Embryonic Chick or Quail Dermal Fibroblasts through Cell-Cell Interaction: Correlation of a Transient Increase in Retinoic Acid Receptor β mRNA in Retinol-Treated Dermal Fibroblasts with Their Competence to Induce Epidermal Mucous Metaplasia

Epidermal mucous metaplasia of 13-day-old chick embryonic tarsometatarsal skin can be induced by culture in medium containing 20 μM retinol for only 8 hr and then in a chemically defined medium without retinol for 2 days. Retinol primarily affects the dermal cells, which then transform the epithelial cells into mucus-secreting cells. In this study, we developed a system using a combination of retinol-pretreated chick or quail dermal fibroblasts and chick skin, and showed that retinol-pretreated quail embryonic dermal fibroblasts invaded the dermis of chick embryonic skin to beneath the epidermal basal cells within 1 day of culture and induced metaplasia, suggesting that epidermal mucous metaplasia of the skin was induced by the direct interaction of retinol-pretreated dermal fibroblasts with the epidermal cells or by low diffusible paracrine factor produced by the fibroblasts.
Increase in retinoic acid receptor β (RARβ) mRNA in dermal fibroblasts was observed after 8 hr-treatment with retinol which preceded morphological changes induced by retinol and this increase was correlated with the competence of the dermal fibroblasts to induce epidermal mucous metaplasia. Thus some gene product(s) controlled by RARβ in dermal fibroblasts may be an essential signal for induction of epidermal mucous metaplasia.     

Short term retinol treatment in vitro induces stable transdifferentiation of chick epidermal cells into mucus-secreting cells

Summary Epidermal mucous metaplasia of cultured skin can be induced by treatment with excess retinol for several days (Fell 1957). In the induction of mucous metaplasia, retinol primarily affects the dermal cells and retinol-pretreated dermis can alter epidermal differentiation towards secretory epithelium (Obinata et al. 1987). In this work, we found that mucous metaplasia could be induced by culturing 13-day-old chick embryonic tarsometatarsal skin in medium containing retinol (20 M) for only 8–24 h, followed by culture in a chemically defined medium (BGJb) without retinol or serum for 6 days. The application of cycloheximide together with retinol during the first 8 h of culture inhibited epidermal mucous metaplasia during subsequent culture for 6 days in BGJb, indicating that induction of a signal(s) in the dermis by excess retinol requires protein synthesis. However, the presence of 20 nM hydrocortisone (Takata et al. 1981) throughout the culture period did not inhibit retinol-induced epidermal mucous metaplasia of the epidermis. This indicates that a brief treatment of the skin with excess retinol determines the direction of epithelial differentiation toward secretory epithelium; this is a simpler in vitro system for the induction of epidermal mucous metaplasia than those established before. Offprint requests to: A. Obinata     

Acceleration of Retinol-Induced Epidermal Mucous Metaplasia by Stimulating the Dermal Adenylate Cyclase-cAMP System in Chick Embryonic Skin: Appearance of cAMP-Dependent Phosphorylated Proteins in Dermis of Retinol-Pretreated Skin after 2 h-Treatment with cAMP

Epidermal mucous metaplasia of cultured 13-day-old chick embryonic tarsometatarsal skin can be induced by culture in medium containing excess retinol (20 μM) for only 8–24 h and then in a chemically defined medium with Bt₂cAMP (0.2–2 mM) and without retinoids or serum for 2 days. In this work, stimulation of the adenylate cyclase-cAMP system in retinol-pretreated skin by forskolin, pertussis toxin, cholera toxin or AIF₄^– was found to accelerate the synthesis of epidermal sulfated glycoprotein (mucin). In skin induced toward mucous metaplasia by retinol, treatment with forskolin for 1 day increased the cAMP content 10-fold in the dermis but only 2-fold in the epidermis over the control levels. The cAMP level of Bt₂cAMP (0.2 mM)-treated skin was 18 times higher in the dermis but rather lower in the epidermis than untreated skin. These results suggest the importance of an adenylate cyclase-cAMP system in the dermis of skin in stimulating mucous metaplasia induced by retinoids. In fact, cAMP-dependent protein phosphorylation was seen only in the dermis of retinol-pretreated skin after 2 h-treatment with cAMP. As no transfer of cAMP from the dermis to the epidermis of forskolin-treated skin was detected, there may be no gap junctional communication between the epidermis and the dermis, while the basement membrane becomes discontinuous during mucous metaplasia.     

Induction of epidermal mucous metaplasia by culture of recombinants of undifferentiated epidermis and retinol-treated dermis in a chemically defined medium

Epidermal mucous metaplasia of cultured skin is known to be induced by excess retinol. Studies were made on whether retinol affects primarily the epidermis or the dermis during retinol-induced epidermal mucous metaplasia of 13-day-old chick embryonic skin in culture. When recombinants of 13-day-old normal epidermis and retinol-treated dermis were cultured for 7 days in chemically defined medium in the absence of retinol, hormones, and serum, they showed altered epidermal differentiation toward secretory epithelium (mucous metaplasia). Thus retinol acted primarily on dermal cells.     

Inhibition by epidermal growth factor (EGF) of epidermal DNA synthesis in cultured chick embryonic skin pretreated with retinol and/or hydrocortisone: specific increment in EGF binding activity in both retinol- and hydrocortisone-pretreated epidermis without correlation to EGF-mediated inhibition of cell growth.

When tarsometatarsal skin of 13-day-old chick embryos that had been cultured in medium containing 5% delipidized FCS with or without retinol (20 microM) and/or hydrocortisone (20 nM) for 1 day was cultured in a chemically defined medium without either the hormone or retinol for 1 day, epidermal DNA synthesis of hydrocortisone- and/or retinol-pretreated skin was inhibited when compared to that of control skin. The addition of epidermal growth factor (EGF, 10 ng/ml) to retinol- or hydrocortisone-pretreated skin further inhibited the epidermal DNA synthesis. Epidermal DNA synthesis in retinol- and hydrocortisone-pretreated skin was more strongly inhibited than in retinol- or hydrocortisone-pretreated skin, but was not further inhibited by EGF. In epidermis which was induced to differentiation toward keratinization by hydrocortisone or mucous metaplasia by retinol, EGF inhibited DNA synthesis. The extent of [125I]-EGF binding to the epidermis of retinol- and hydrocortisone-pretreated skin was 160-180% that in control skin, with no change in affinity. Hence there is no correlation between EGF-binding and the mitogenic activity of EGF.     

Increase in expression of the homeobox gene, GBX1, in retinol-induced epidermal mucous metaplasia

Using a degenerate RT-PCR-based screening method, we isolated the homeobox gene, Gbx1, from the shank skin of 13-day-old chick embryos. By in situ hybridization analysis we showed that the Gbx1 was expressed in the epidermis of the skin and the mucous epithelium of the intestine, and that among many homeobox genes isolated, expression of the Gbx1 strongly increased in the epidermis when the skin was cultured with 20 microM retinol, which induces epidermal mucous metaplasia. The Gbx1 expression in the epidermis was increased by interaction with the retinol-pretreated dermal fibroblasts, resulting in mucous metaplasia. These results suggest that the Gbx1 regulates the differentiation and transdifferentiation of the epithelium and controls the morphology of the epithelium. We isolated the chick Gbx1 cDNA clones. The amino acid sequences in homeodomain and its downstream encoded by human and chick Gbx1 cDNA were almost the same, but those upstream of the homeodomain were rather different.     

CRH functions as a growth factor/cytokine in the skin

We tested the effect of CRH and related peptides in a large panel of human skin cells for growth factor/cytokine activities. In skin cells CRH action is mediated by CRH-R1, a subject to posttranslational modification with expression of alternatively spliced isoforms. Activation of CRH-R1 induced generation of both cAMP and IP3 in the majority of epidermal and dermal cells (except for normal keratinocytes and one melanoma line), indicating cell type-dependent coupling to signal transduction pathways. Phenotypic effects on cell proliferation were however dependent on both cell type and nutrition conditions. Specifically, CRH stimulated dermal fibroblasts proliferation, by increasing transition from G1/0 to the S phase, while in keratinocytes CRH inhibited cell proliferation. In normal and immortalized melanocytes CRH effect showed dichotomy and thus, it inhibited melanocyte proliferation in serum-containing medium CRH through G2 arrest, while serum free media led instead to CRH enhanced DNA synthesis (through increased transition from G1/G0 to S phase and decreased subG1 signal, indicating DNA degradation). CRH also induced inhibition of early and late apoptosis in the same cells, demonstrated by analysis with the annexin V stains. Thus, CRH acts on epidermal melanocytes as a survival factor under the stress of starvation (anti-apoptotic) as well as inhibitor of growth factors induced cell proliferation. In conclusion, CRH and related peptides can couple CRH-R1 to any of diverse signal transduction pathways; they also regulate cell viability and proliferation in cell type and growth condition-dependent manners.     

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. II. A histomorphological study

The effects of all-trans retinol and cigarette smoke condensate (CSC) on tissue morphology and cellular differentiation were investigated in vitamin A-deprived tracheal epithelium cultured in vitamin A-and serum-free hormone-supplemented medium. Physiological retinol concentrations prevented the development of hyperplasia and squamous metaplasia with or without keratinization, and induced differentiation to mucous cells. Squamous metaplastic foci with keratinization were observed during 12 days of culture with low retinol concentrations and with dimethylsulfoxide (DMSO) which was accompanied by an increased number of basal and indeterminate cells. CSC induced a dose-related hyperplasia and irregularly shaped foci of squamous metaplasia with atypical epithelial proliferation. In non-metaplastic epithelium, CSC exposure increased the number of ciliated cells. Hyperplasia and squamous metaplasia were inhibited if the tracheal rings were first treated with retinol followed by CSC exposure, or if the tracheas were simultaneously treated with retinol and CSC. CSC-exposure prior to retinol treatment induced similar histomorphological alterations as CSC alone.     

Modulation of hepatocyte growth factor induction in human skin fibroblasts by retinoic acid

Topical treatment of skin with all-trans-retinoic acid (ATRA), the major biologically active form of vitamin A, results in hyperproliferation of basal keratinocytes, leading to an accelerated turnover of epidermis cells and thickening of the epidermis, probably via induction of production of paracrine growth factors for keratinocytes in epidermal suprabasal keratinocytes and/or dermal fibroblasts. Since hepatocyte growth factor (HGF) is a factor mitogenic to epidermal keratinocytes secreted from dermal fibroblasts, the effect of ATRA on basal and induced HGF production in human dermal fibroblasts in culture was examined. ATRA alone did not induce HGF production, but it significantly enhanced HGF production induced by the cAMP-elevating agent cholera toxin or the membrane-permeable cAMP analog 8-bromo-cAMP. Cholera toxin-induced activation of cAMP responsive element (CRE)-binding protein (CREB) was enhanced by pretreating cells with ATRA for 24 h. In contrast, HGF production induced by epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) was potently inhibited by ATRA. These modulatory effects of ATRA were different from the effects of transforming growth factor-beta1 (TGF-beta) and dexamethasone, both of which inhibited HGF production induced by all of the four inducers. Up-regulation of HGF gene expression by cholera toxin and EGF was also enhanced and inhibited, respectively, by ATRA. Both 9-cis-retinoic acid (9-cis-RA) and 13-cis-retinoic acid (13-cis-RA), which are stereo-isomers of ATRA, showed a modulatory effect on HGF induction similar to that of ATRA. These results suggest that ATRA augments the induction of HGF production caused by increased intracellular cAMP.     

Inhibition of endothelial secretion of tissue-type plasminogen activator and its rapid inhibitor by agents which increase intracellular cyclic AMP

We investigated the effect of agents which raise intracellular levels of cyclic AMP (cAMP) on the secretion of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured human umbilical-vein endothelial cells. Significant inhibition of baseline (unstimulated) t-PA and PAI-1 secretion was observed in response to several agents which, when added exogenously, cause increased intracellular cAMP: cholera toxin, 1-methyl-3-isobutylxanthine (MIX), dibutyryl-cAMP, and prostaglandin E1. These agents also significantly reduced or abolished the previously reported stimulatory effects of thrombin and histamine on t-PA secretion, and, with the exception of MIX, significantly reduced the previously reported stimulatory effect of thrombin on PAI-1 secretion. MIX at a concentration (10 microM) below that required to inhibit t-PA and PAI-1 secretion when tested alone, significantly increased the inhibitory effects of cholera toxin, dibutyryl-cAMP, and prostaglandin E1 on both t-PA and PAI-1 secretion. The data suggest that elevated intracellular levels of cAMP inhibit both spontaneous endothelial secretion of t-PA and PAI-1, and secretion induced by agents (thrombin and histamine) which stimulate endothelial phosphoinositide metabolism, consistent with bidirectional regulation of endothelial fibrinolytic protein secretion by the adenylate cyclase and phosphoinositide signal transduction pathways. The inhibitory effects of cAMP do not appear to be specific for t-PA and PAI-1, since cholera toxin and MIX also inhibited endothelial secretion of the adhesive protein, fibronectin. Significant inhibition of baseline endothelial t-PA and PAI-1 secretion was also caused by the stable prostacyclin analogue iloprost (ZK 36 374) and by arachidonic acid, which is converted by endothelial cells to prostacyclin, suggesting that prostacyclin produced endogenously by endothelial cells may inhibit secretion of fibrinolytic proteins by increasing intracellular cAMP.     

Differential inhibition of T cell receptor signal transduction and early activation events by a selective inhibitor of protein-tyrosine kinase

Engagement of the TCR (CD3-Ti) by Ag/MHC, CD3 mAb, or lectin mitogen stimulates the very early tyrosine phosphorylation of several cellular substrates including TCR-zeta. The T cell specific protein-tyrosine kinase (PTK), p56lck, has been implicated in the tyrosine phosphorylation of TCR-zeta. However, the significance of this event with regard to CD3-Ti signal transduction remains unclear. Herein, we have investigated the effect of the selective PTK inhibitor genistein (4',5,7-trihydroxyisoflavone) on cellular events associated with activation via CD3-Ti triggering. Genistein inhibited the T cell PTK, p56lck, in a dose-dependent fashion with an ID50 = 40 microM. Genistein also inhibited CD3 mAb or PHA-induced TCR-zeta chain phosphorylation in intact peripheral blood T cells. Genistein blocked the expression of IL-2 and IL-2R (CD25) in T cells stimulated with PHA/PMA or CD3 mAb/PMA, but did not inhibit the de novo expression of the CD69 early activation Ag, which is induced primarily by a PKC-dependent pathway. IL-2 and CD25 expression induced by calcium ionophore A23187 and PMA was largely refractory to inhibition by genistein, suggesting an effect of the drug on calcium-dependent pathways stimulated via CD3-Ti triggering. In this last regard, genistein partially inhibited the CD3 mAb-induced rise in [Ca2+]i but did not inhibit PHA- or CD3 mAb-induced phosphatidylinositol hydrolysis. Consequently, protein-tyrosine phosphorylation does not appear to be a prerequisite for CD3-Ti-mediated activation of phosphatidylinositol-specific phospholipase C activity and PIP2 hydrolysis. An alternative role for PTK in CD3-Ti signal transduction is suggested.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Regulation of the respiratory burst by cyclic 3',5'-AMP, an association with inhibition of arachidonic acid release.

The mechanism of cAMP regulation of the respiratory burst was studied with HL-60 cells that had been DMSO-differentiated to a neutrophil-like cell. To evaluate the effects of known cAMP concentrations, cells were permeabilized with streptolysin-O. Chemotactic peptide (FMLP)-stimulated NADPH oxidase activity was inhibited by cAMP at concentrations higher than 3 microM. Because intracellular calcium was buffered, inhibitory actions of cAMP were not mediated by modulation of calcium concentration. Effects of cAMP on chemotactic peptide signal transduction mediated by phospholipase C, phospholipase D, and phospholipase A2 were then determined. Neither inositol phosphate generation (phospholipase C) nor phosphatidylethanol generation (phospholipase D activity in presence of 1.6% ethanol) induced by FMLP were significantly affected by cAMP. In contrast, cAMP potently inhibited FMLP-induced arachidonic acid mobilization (phospholipase A2). NADPH oxidase activity induced by exogenous arachidonic acid was not inhibited by cAMP. These results indicate that cAMP-mediated inhibition of arachidonic acid mobilization may be important in regulation of the respiratory burst.     

Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE2

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 microM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 microM; an inhibitor of the EP1-EP4 prostaglandin receptors) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 microM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE(2) (1 microM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). ACh and ionomycin increased PGE(2) release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [Ca(2+)](i) activates Ca(2+)-regulated exocytotic events and PGE(2) release mediated by COX-1. The released PGE(2) induces the accumulation of cAMP, which enhances the Ca(2+)-regulated exocytosis. The autocrine mechanism mediated by PGE(2) maintains the high-level mucin release from antral mucous cells during ACh stimulation.     

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.     

Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H(+)-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator N(6)-monobutyryl-3'-5'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 microM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 microM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 microM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.     

Inhibitory effect of 17 beta -estradiol on prostaglandin E2-induced phosphoinositide hydrolysis in osteoblast-like cells.

We examined the effect of estradiol on PGE2-induced phosphoinositide hydrolysis and cAMP production in cloned osteoblast-like MC3T3-E1 cells. 17 beta -Estradiol pretreatment significantly inhibited the formation of inositol phosphates induced by 10 microM PGE2 in a dose-dependent manner between 1 pM and 10 nM. This effect of 17 beta -estradiol was dependent on the time of pretreatment and submaximum inhibition was observed at 4 h. However, 17 beta -estradiol had little effect on the formation of inositol phosphates induced by 20 mM NaF, a GTP-binding protein activator. The cAMP production induced by PGE2 was not influenced by 17 beta -estradiol. These results suggest that 17 beta -estradiol modulates the signal transduction by PGE2 and that the effect seems to be exerted between PGE2 receptor and the GTP-binding protein coupled to phospholipase C in osteoblast-like MC3T3-E1 cells.     

Effects and interactions between alpha-MSH and MCH/NEI upon striatal cAMP levels.

It is known that alpha-MSH augments cAMP levels in rat brain slices containing accumbens and caudate-putamen nuclei. In this study we examined: a) the effect of other neuropeptides: MCH and NEI, on this cyclic nucleotide; b) if the effects of alpha-MSH on cAMP production can be modulated by addition of MCH or NEI to the incubation medium. Both MCH and NEI (3.6 microM) increased the production of cAMP, whereas at doses of 0.6 microM exerted no effects. When alpha-MSH 0.6 microM was added with NEI or MCH (0.6 microM), only MCH blocked the increase in the cAMP induced by alpha-MSH. Neither MCH nor NEI at the highest dose used (3.6 microM) had any additive effect on AMPc when added together with alpha-MSH. We conclude that, at a high concentration, (MCH/NEI)-like peptides can use the intracellular signal transduction linked to cyclic nucleotides in the CNS.     

Protein kinase C mediates induced secretion of vascular endothelial growth factor by human glioma cells

To understand how vascular endothelial growth factor (VEGF) production is activated in malignant glioma cells, we employed protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors to evaluate the extent to which these protein kinases were involved in signal transduction leading to VEGF production. PTK inhibitors blocked glioma proliferation and epidermal growth factor (EGF)-induced VEGF secretion, while H-7, a PKC inhibitor, inhibited both EGF-induced and baseline VEGF secretion. Phorbol 12-myristate 13-acetate (PMA), a non-specific activator of PKC, induced VEGF secretion by glioma cells, which was enhanced by calcium ionophore A23187, but completely blocked after prolonged treatment of cells with 1 microM PMA, by presumably depleting PKC. All inhibitors (genistein, AG18, AG213, H-7, prolonged PMA treatment) which inhibited EGF-induced VEGF secretion in glioma cells also inhibited cell proliferation at similar concentrations. However, PKC inhibition only blocked 50% of the VEGF secretion induced by growth factors (EGF, platelet-derived growth factor-BB, or basic fibroblast growth factor). This reserve capacity could be ascribed to a PKC-independent effect, or to PKC isoenzymes not down-regulated by PMA. These findings extend our previous assertion that VEGF secretion is tightly coupled with proliferation by suggesting that activation of convergent growth factor signaling pathways will lead to increased glioma VEGF secretion. Understanding of signal transduction of growth factor-induced VEGF secretion should provide a rational basis for the development of novel strategies for therapy.     

Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.