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A comparison of the production of tissue-type plasminogen activator (t-PA) and gelatinases A and B was made at the mRNA and protein levels in human Bowes melanoma cells treated with phorbol myristate acetate (PMA). Immunocytochemical analysis confirmed previous quantitative data on PMA-mediated induction of t-PA. It also showed that t-PA immunoreactivity can be restrained to the local environment of the producing cell, most probably by interaction with extracellular matrix components. Zymographical analysis showed that gelatinase B protein was induced by PMA, whereas gelatinase A remained at the constitutive level. Protein kinase C (PKC) appeared to be involved in this regulation since, after PMA treatment (1) the PKC activity was found to be translocated from the cytosol to the particulate fraction of the cells and (2) addition of staurosporine and H-7 blocked the gelatinase B increase. Northern-blot hybridization showed a transient rise in t-PA and gelatinase B mRNA levels whereas gelatinase A mRNA levels remained unchanged. When c-fos and c-jun mRNAs were investigated, only that of c-fos was affected by PMA. Activation by PMA can be kinetically ordered as follows: translocation of PKC to the membrane fraction, transcription of the c-fos gene and eclipsing of gelatinase B mRNA, increase in steady-state mRNA levels of t-PA and gelatinase B and, finally, secretion of t-PA and gelatinase B glycoproteins. Our data also suggest that various proteases that are known to cooperate in the remodeling of the extracellular matrix can be differently regulated in one tumor-cell type.

Treatment of isolated rat adipocytes with tumor-promoting phorbol esters, caused a fivefold stimulation of glucose oxidation, determined as 14C0, production from [l-'4C]glucose and a fivefold increase in the rate of lipid synthesis from [ ''C]glucose. Treatment of the cells with 12-0-tetradecanoylphorbol 13-acetate increased the rate of 86Rbf uptake into the cells. Also phospholipase C was able to stimulate the rate of glucose oxidation; phospholipase C and 12-0-tetradecanoylphorbol 13-acetate stimulated glucose oxidation in a non-synergistic fashion, indicating a common mechanism for their action. Active phorbol esters and, in part, also phospholipase C, caused a translocation of protein kinase C activity from the soluble to the particulate fraction of the adipocytes. This process was rapid, being complete 30 s after the addition of phorbol ester, and resulted in the appearance of the kinase mainly in the mitochondria1 and plasma membrane fractions. A comparison between the binding characteristics of adipocyte protein kinase C and the metabolic effects of the phorbol esters on the adipocytes revealed that the dose-response relationship did not correlate with binding of the phorbol esters, but, rather, a correlation was observed between the dose of phorbol esters required for translocation of protein kinase C and the intracellular effects. The results indicate that the intracellular translocation of protein kinase C might be a trigger for the effects of phorbol esters on the adipocyte and that binding of the esters to protein kinase C is not a sufficient event to cause this effect. Furthermore, it is suggested that activation of protein kinase C might be partly the action of hormones, such as insulin, on the fat cells.The adipocyte is characterized by its hormone sensitivity. Numerous hormonal responses have been identified and many events following the hormone binding to the cell surface are apparently mediated by CAMP-dependent kinases. The demonstration of a calcium-dependent and phospholipid-dependent protein kinase (protein kinase C) first in brain [l] and subsequently in many other tissues [2, 31, including adipose tissue [4], raises the question to what extent does this type of kinase participate in the hormonal influence on the metabolic control in adipocytes. It has been found that the activity of protein kinase C in isolated adipocytes is comparable to the level of activity of CAMP-dependent protein kinase [4]. This finding prompted us to investigate any roles of protein kinase C in the adipocyte.Accumulating evidence suggest that agents, including hormones, that stimulate phosphatidylinositol turnover leading to diacylglycerol production [5,6] also activate protein kinase C. Diacylglycerol apparently decreases the requirement of the enzyme for calcium and phospholipid [7]. Furthermore, exposure of adipose tissue to phospholipase C, generating diacylglycerols by phospholipid hydrolysis, has been shown to elicit various metabolic effects similar to those produced by insulin [8 -101. Phorbol esters can ...

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