In serum-starved NIH 3T3 clone 7 fibroblasts, choline phosphate (ChoP) (0.5-1 mM) and insulin synergistically stimulate DNA synthesis. Here we report that ATP also greatly enhanced the mitogenic effects of ChoP (0.1-1 mM) both in the absence and presence of insulin; maximal potentiating effects required 50 -100 M ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5-O-(3-thiotriphosphate), adenosine 5-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indicating the mediatory role of a purinergic P 2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibroblasts, indicating that ChoP can be a mitogen only if it is released from cells. Extracellular ATP (10 -100 M) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on the activity state of p42/p44 mitogen-activated protein kinases, while in combination these agents stimulated the activity of phosphatidylinositol 3-kinase (PI 3-kinase). Expression of a dominant negative mutant of the p85 subunit of PI 3-kinase or treatments with the PI 3-kinase inhibitor wortmannin only partially (ϳ40 -50%) reduced the combined effects of ChoP, ATP, and insulin on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost completely inhibited these effects. ATP and insulin also potentiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective concentrations of ChoP and S1P synergistically stimulated DNA synthesis. The results indicate that in the presence of extracellular ATP and/or S1P, ChoP induces mitogenesis through an extracellular site by mechanisms involving the activation of pp70 S6 kinase and, to a lesser extent, PI 3-kinase.

Human placental alkaline phosphatase (PALP) is synthesized in the placenta during pregnancy and is also expressed in many cancer patients; however, its physiological role is unknown. Here we show that in human fetus fibroblasts as well as normal and H-ras-transformed mouse embryo fibroblasts PALP stimulates DNA synthesis and cell proliferation in synergism with insulin, zinc and calcium. The mitogenic effects of PALP are associated with the activation of c-Raf-1, p42/p44 mitogen-activated protein kinases, p70 S6 kinase, Akt/PKB kinase and phosphatidylinositol 3P P-kinase. The results suggest that in vivo PALP may promote fetus development as well as the growth of cancer cells which express oncogenic Ras.z 2000 Federation of European Biochemical Societies.

In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mm Ca 2+ -containing medium, addition of 0.75±2 mm extra Ca 2+ stimulated DNA synthesis in synergism with zinc (15±60 mm), insulin and insulin-like growth factor I. Extra Ca 2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca 2+ -induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca 2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca 2+ effects, cotreatments with the choline kinase substrate choline (250 mm) and the choline kinase inhibitor hemicholinium-3 (2 mm) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca 2+ (3.8 mm total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1±2 mm extra Ca 2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca 2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca 2+ in the dermal layer, Ca 2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors.Keywords: calcium; zinc; insulin; DNA synthesis.Our work is aimed at determining the role of extracellular Ca 2+ in the regulation of fibroblast growth during wound healing. While the concentration of Ca 2+ in the circulation is maintained in the 1.1±1.3 mm range [1,2], several cellular systems [3±6], including fibroblasts [7], have been shown to express specific cell surface receptors which respond to 2±4 mm concentrations of extracellular Ca 2+ . Importantly, the concentration of Ca 2+ in the dermal layer is in the range that can potentially activate Ca 2+ receptors [8], and which have been shown to stimulate dermal fibroblast growth [9]. Apart from the findings that the mitogenic actions of Ca 2+ involve the activation of the p42/p44 mitogen-activated protein (MAP) kinases [7,9], relatively little is known about the mechanism of growth regulatory effects of Ca 2+ . For example, no data are available concerning the potential interactions between Ca 2+ and the numerous factors that may be involved in the stimulation of fibroblast growth in wounded tissues. It has been well established that zinc is an important micronutrient required for normal growth and many other biological functions [10±14]. Zinc influences, or is required for, the function of many enzymes as well as several transcription factors, and zinc deficiency results in delayed wound healing among many other symptoms [11±14]. P...