Abstract
Taking advantage of the potent mitogenic effect exerted by insulin in human colonic cells, we used Caco-2 cells transfected with an activated (Val-12) human Haras gene or the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity, to investigate the effect of oncogenic p21ras and PyMT/pp60c-src on insulin mitogenic signaling. As compared to vector control Caco-2 cells, both oncogene-transfected cells exhibited: 1) a loss of response to insulin's stimulatory effect on mitogen-activated protein (MAP) kinase activity and cell proliferation, both of which were constitutively increased; 2) a decrease in insulin receptor (IR) affinity and insulin-stimulated exogenous tyrosine kinase activity, which resulted from increased protein kinase C (PKC) activity (Delage, S., Chastre, E., Empereur, S., Wicek, D., Veissiére, D., Capeau, J., Gespach, C., and Cherqui, G. (1993) Cancer Res. 53, 2762-2770), since IR alterations were corrected by PKC down-regulation; and 3) a decrease in both IR mRNA level and IR number, which was independent of PKC since it persisted after PKC down-regulation. In conclusion, this is the first evidence that oncogenic p21ras and PyMT/pp60c-src abolish insulin mitogenic signaling in human colonic cells through mechanisms involving (i) constitutive activation of MAP kinase and (ii) marked decreases in both IR function and expression which are mediated by PKC-dependent and PKC-independent pathways, respectively.
Highlights
Taking advantage of the potent mitogenic effect ex- factor (8-10)or insulin-like growth factor (IGF-I)' (11).These erted by insulin in human colonic cells, we used Caco-2 cells transfected with an activated (Val-12) human Har m gene or the polyoma middle T (PyMT) oncogene, a constitutive activatorof pp60c'mtyrosinekinase activity, to investigate the effect of oncogenic p21" and PyMT/ pp60c-""oninsulin mitogenic signaling
As compared to vector control Caco-2 cells, both oncogene-transfected cells exhibited: 1) a loss of response to insulin's stimulatory effect on mitogen-activatedprotein ( M A P ) kinase activity and cell proliferation, both of which were constitutively increased; 2j a decrease in insulin receptor (IR) affinity and insulin-stimulatedexogenous tyrosine kinase activity, which resulted from increased protein kinase C (PKC) activity (Delage, S., Chastre, E., Empereur, s., Wicek, D., VeissiBre, D., Capeau, J., Gespach, C., andCherqui, G. (1993) Cancer Res. 53,2762-2770), since IR alterations were corrected by PKC down-regualterations were shown t o occur at receptor andor post-receptor levels and, most important, to result in different effects on cellular growthresponses according to thegrowth factor examined
Number, which was independent of PKC since it per- Human colonic epithelial cells have been shown to express sisted after PKC down-regulation.In conclusion, this is IRs (20, 21) and to be highly responsive to insulin's mitogenic the first evidence that oncogenic p21" and PyMT/ effect (20, 22)
Summary
Caco-2 cells were seeded (day1)in 30-cm2plates (4or 8 x lo5cells/plate) 60-cm2plates were washed with phosphate-freeDMEM and incubated and cultured iDn MEM supplemented with5%fetal calf serum for 48 h for 16h in 4 ml ofthe same medium conta5in0i0npgCi of [32Plorthophosand in serum-free medium for 24 h. Binding Assays-Serum-deprived monolayers of transfected Caco-2 antibodies coupled to the rabbit anti-rat IgGProtein A-Sepharosceomcells which had been treated for the indicated times with or withoutplethxe. Immunoprecipitates werecollected and washed eight times with 50 indicated concentrations of PMA were incubated a t 15 "C in 0.5 ml of mM Hepes buffer, pH 7.4,500mM NaCl, 5mM MgCl,, 0.1% TritonX-100, Krebs-Ringer phosphaternepes buffer, 1% fattyacid-free BSA, pH 7.5, 0.005% SDS. Insulin binding to whole cells in suspensionor to WGA-purified receptors
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