We have shown that hyaluronic acid stimulates the proliferation of quiescent NIH 3T3 cells. We have shown that treatment of 1 mg/ml hyaluronic acid results in increase of tyrosine phosphorylation of two proteins, MW 124 kDa and 60 kDa as detected by anti-tyrosine antibodies by Western blot analysis. Maximum phosphorylation occurred within 2 h after addition of 1 mg/ml hyaluronic acid. Stimulation of proliferation was also accompanied by increase in c-Myc protein, which was inhibited by amlloride, an inhibitor of Na+/H+ antiporter and EGTA and increase in the steady state level of pRb, the RB gene product. These results suggest that the intracellular signal transduction pathways that mediate the stimulatory effects of hyaluronic acid on cellular proliferation are similar to those of growth factors.
3T3 Cells ; Amiloride/pharmacology ; Animal ; Cell Division ; Dose-Response Relationship, Drug ; Egtazic Acid/pharmacology ; Hyaluronic Acid/pharmacology* ; Mice ; Mitogens/pharmacology* ; Phosphoproteins/metabolism* ; Phosphorylation ; Proto-Oncogene Proteins c-myc/metabolism* ; Retinoblastoma Protein/metabolism* ; Signal Transduction ; Sodium-Hydrogen Antiporter/antagonists & inhibitors ; Tyrosine

Both adaptive and deleterious responses of cells to ethanol are likely triggered by short-term interactions of the cells with ethanol. Many studies have demonstrated the direct effect of ethanol on growth factor-stimulated cell proliferation. Using Swiss 3T3 cells whose growth was inhibited by ethanol in a concentration-dependent manner, we further investigated the molecular mechanisms of acute ethanol treatment by examining its effect on EGF- and PDGF-mediated cellular signaling systems for the mitogenic function. Tyrosine autophosphorylation of the growth factor receptors was partially prevented by ethanol in intact cells. When ethanol was included before or after EGF stimulation, no effect on the receptor signaling was observed. Here we also report that ethanol inhibits activation of ERK induced by both EGF and PDGF. EGF-induced JNK activation was reduced but PDGF-induced rapid JNK activation was delayed by the addition of ethanol. The balance between its inhibitory and stimulatory effect on the signaling molecules might determine the rate of cell growth.
3T3 Cells ; Animal ; Cell Division/drug effects ; Drug Interactions ; Epidermal Growth Factor/pharmacology* ; Ethanol/pharmacology* ; Mice ; Mitogen-Activated Protein Kinases/metabolism ; Mitogens/pharmacology* ; Platelet-Derived Growth Factor/pharmacology* ; Signal Transduction/drug effects

Phospholipase Cgamma1 (PLCgamma1) plays an important role in controlling cellular proliferation and differentiation. PLCgamma1 is overexpressed in some tumors, and its overexpression induces solid tumors in nude mice. However, the regulatory mechanisms underlying PLCgamma1-induced cell proliferation are not fully understood. Here we show that overexpression of PLCgamma1 highly phosphorylated glycogen synthase kinase-3beta (GSK-3beta) at serine-9 in 3Y1 fibroblasts. Inhibition of protein kinase C (PKC)s with GF109203X abrogated GSK-3beta phosphorylation by PLCgamma1. We also found that steady-state level of cyclin D1 protein, but not cyclin D1 mRNA, was highly elevated in response to serum stimulation in PLCgamma1-transfected cells as compared with vector-transfected cells. Since GSK-3beta is involved in cyclin D1 proteolysis in response to mitogenic stimulation, PLCgamma1-mediated GSK-3beta phosphorylation may function as a regulation of cyclin D1 accumulation in PLCgamma1-overexpressing cells.
Animals ; Cyclin D1/metabolism ; Epidermal Growth Factor/pharmacology ; Fibroblasts ; Gene Expression ; Glycogen Synthase Kinase 3/chemistry/*metabolism ; Mitogens/pharmacology ; Phospholipase C/genetics/*metabolism ; Phosphorylation/drug effects ; Phosphoserine/*metabolism ; Protein Kinase C/antagonists & inhibitors/*metabolism ; Rats ; Signal Transduction

BACKGROUNDHuman urotensin II (UII) is the most potent mammalian vasoconstrictor identified so far. Our previous study showed that UII is a potent mitogen of airway smooth muscle cells (ASMC) inducing ASMC proliferation in a dose-dependent manner. The signal transduction pathway of UII mitogenic effect remains to be clarified. This study was conducted to investigate the signal transduction pathway in the proliferation of ASMC induced by UII.

METHODSIn primary cultures of rat ASMCs, activities of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and calcineurin (CaN) induced by UII were measured. The effect of CaN on PKC and MAPK was studied by adding cyclosporin A (CsA), a specific inhibitor of CaN. Using H7 and PD98059, inhibitors of PKC and MAPK, respectively, to study the effect of PKC and MAPK on CaN. The cytosolic free calcium concentration induced by UII was measured using Fura-2/AM.

RESULTSUII 10(-7) mol/L stimulated ASMC PKC and MAPK activities by 44% and 24% (P < 0.01), respectively, after incubating for 20 minutes. It increased CaN activity in a time-dependent manner, being 1.68 times as that of control for 24 hours (P < 0.01). It promoted the cytosolic free calcium concentration increase of 18% (P < 0.01). CsA 10(-6) mol/L and H7 50 micromol/L inhibited UII-stimulated CaN activity by 45% (P < 0.01) and 21% (P < 0.05), respectively, while PD98059 50 micromol/L had no effect on CaN activity (P > 0.05). CsA 10(-6) mol/L inhibited UII-stimulated PKC activity by 14% (P < 0.05), while having no effect on MAPK activity (P > 0.05).

CONCLUSIONSUII increases cytosolic free calcium concentration and activates PKC, MAPK and CaN. The signal transduction pathway between PKC and CaN has cross-talk.

Animals ; Calcineurin ; metabolism ; Cells, Cultured ; Enzyme Activation ; Mitogen-Activated Protein Kinases ; metabolism ; Mitogens ; pharmacology ; Myocytes, Smooth Muscle ; cytology ; Protein Kinase C ; metabolism ; Rats ; Signal Transduction ; physiology ; Trachea ; cytology ; Urotensins ; pharmacology

The studies were conducted on Balb/c mice exposed to restraint stress twice for 12 h at 24 h intervals. Prior to restraint stress the mice were treated with sodium diethyldithiocarbamate (DTC) i.p. at a dose of 20 mg/kg five times at 48 h intervals. DTC was used per se or with zinc ions interaction, by adding zinc sulfate to drinking water at a dose of 72 microgram/mouse daily. The results obtained in the study show that restraint stress causes involution of lymphatic organs, decreased the percentage of immature (CD4+CD8+) and, mature (CD4+) thymocytes and CD4+, CD8+and CD19 + splenocytes and proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohemagglutinin (PHA). The restraint stress decreased also interleukin-1 (IL-1) production by murine intraperitoneal macrophages stimulated in vitro with lipopolysaccharide (LPS) from E. coli. Pretreatment with DTC counteracted restraint stress-induced immunosuppression, which is expressed as partial normalisation of the total number of thymocytes, splenocytes and IL-1 production, accelerated regeneration of thymus and spleen, shorter suppressive action of restraint stress on the percentage of CD4+CD8+thymocytes and in total normalisation of the CD4+thymocytes and splenocytes. DTC administered prior to restraint stress augmented the proliferative response of thymocytes to two mitogens. The immunocorrecting action of DTC is enhanced by zinc supplementation, expressed in the increased percentage of CD4+thymocytes and splenocytes, CD19 + splenocytes, proliferative activity of thymocytes stimulated with PHA and IL-1 production. The obtained results show that DTC administration can be supplemented with zinc in order to restore the immune system impaired by stress.
Adjuvants, Immunologic/*pharmacology ; Animals ; Ditiocarb/*pharmacology ; Female ; Immunity, Cellular/*drug effects ; Interleukin-1/biosynthesis ; Macrophages, Peritoneal/immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mitogens/biosynthesis ; Organ Size/drug effects ; Restraint, Physical ; Spleen/cytology/drug effects ; Stress/etiology/*immunology ; T-Lymphocyte Subsets/drug effects ; Thymus Gland/cytology/drug effects ; Zinc Sulfate/*pharmacology

The mechanisms of estrogen and progesterone in human cutaneous pigmentation are largely unknown. The molecular identification of estrogen receptor (ER) and progesterone receptor (PR) in the human melanocytes is of great importance to understand the mechanisms. We performed immunocytochemistry analysis and demonstrated that ER and PR were expressed in the cytoplasms and nuclei of human melanocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis confirmed the expression of ER and PR at the transcriptional level. Despite of the presence of ER and PR, the physiological and pregnant levels of estrogen and progesterone showed inconsistent effects on the proliferation and tyrosinase activity of cultured human melanocytes. These results suggest that human melanocytes express ER and PR, which have a donor-specific action in human pigmentation. Further studies are needed to elucidate the induction mechanism and functions of these receptors, and the role of estrogen and progesterone in melanocytes.
Adult ; Cells, Cultured ; Estrogens/*pharmacology ; Gene Expression ; Humans ; Melanocytes/cytology/*drug effects/metabolism ; Mitogens/pharmacology ; Organ Culture Techniques ; Progesterone/*pharmacology ; Receptors, Estrogen/genetics/*metabolism ; Receptors, Progesterone/genetics/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/drug effects ; Skin Pigmentation/drug effects ; Tissue Donors

The mechanism underlying T cell-mediated fulminant hepatitis is not fully understood. In this study, we investigated whether myeloid derived suppressor cells (MDSCs) could prevent the concanavalin A (ConA)-induced hepatitis through suppressing T cell proliferation. We observed an increase in the frequencies of MDSCs in mouse spleen and liver at early stage of ConA treatment, implicating that the MDSCs might be involved in the initial resistance of mice against ConA-mediated inflammation. Subpopulation analysis showed that the MDSCs in liver of ConA-induced mice were mainly granulocytic MDSCs. Adoptive transfer of the bone marrow-derived MDSCs into ConA-treated mice showed that the MDSCs migrated into the liver and spleen where they suppressed T cell proliferation through ROS pathway. In addition, the frequencies of MDSCs in mice were also significantly increased by the treatment with immune suppressor glucocorticoids. Transfer of MDSCs into the regulatory T cell (Treg)-depleted mice showed that the protective effect of MDSCs on ConA-induced hepatitis is Treg-independent. In conclusion, our results demonstrate that MDSCs possess a direct protective role in T cell-mediated hepatitis, and increasing the frequency of MDSCs by either adoptive transfer or glucocorticoid treatment represents a potential cell-based therapeutic strategy for the acute inflammatory disease.
Adoptive Transfer ; Animals ; Blotting, Western ; Bone Marrow Cells ; immunology ; CD11b Antigen ; immunology ; metabolism ; Cell Movement ; immunology ; Cell Proliferation ; Chemical and Drug Induced Liver Injury ; etiology ; immunology ; prevention & control ; Concanavalin A ; toxicity ; Dexamethasone ; pharmacology ; Flow Cytometry ; Glucocorticoids ; pharmacology ; Liver ; immunology ; pathology ; Male ; Mice, Inbred C57BL ; Mitogens ; administration & dosage ; toxicity ; Myeloid Cells ; immunology ; metabolism ; transplantation ; Receptors, Chemokine ; immunology ; metabolism ; Spleen ; immunology ; pathology ; T-Lymphocytes ; immunology ; T-Lymphocytes, Regulatory ; immunology