Correlations of serum concentrations of 1,25-dihydroxyvitamin D, phosphorus, and parathyroid hormone in tumoral calcinosis.

The inherited metabolic disorder tumoral calcinosis is characterized by elevated serum phosphorus and 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels and paraarticular calcific tumors. The pathogenesis of this disease is obscure, but an elevated renal phosphate reabsorption threshold and increased production of 1,25-(OH)2D are postulated as defects. We studied nine affected patients and found that both serum phosphorus and renal phosphate reabsorption threshold (TmP/GFR) were positively correlated with serum 1,25-(OH)2D levels. Since tumoral calcinosis is a disorder with abnormal renal phosphate transport, we compared the TmP/GFR and serum 1,25-(OH)2D levels to values obtained in patients with two other diseases with renal phosphate transport defects: oncogenic osteomalacia and X-linked hypophosphatemic rickets. We found a significant correlation between TmP/GFR and 1,25-(OH)2D levels in all three diseases, suggesting that in these diseases 1,25-(OH)2D production is regulated in some manner by phosphate transport. Furthermore, previous work indicated that in tumoral calcinosis broad variation exists in serum phosphorus levels. In our patients a negative correlation was found between the serum PTH concentrations and both serum phosphorus levels and TmP/GFR values, respectively. We postulate that although the basic defect in tumoral calcinosis most likely resides in the proximal renal tubular cell, the variation in serum phosphorus levels and possibly disease expression is modulated in part by PTH.

Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens.

Using an N-bromosuccinimide cleavage fragment of histone H1 as a relatively specific substrate for protein kinase C, we evaluated the partitioning of this kinase activity between soluble and particulate cellular fractions in 3T3-L1 fibroblasts. In confluent, serum-deprived cells, protein kinase C activity was approximately equally divided between soluble and detergent-extractable particulate fractions; both rapidly growing and transformed cells appeared to contain higher levels of particulate enzyme activity. Soluble protein kinase C activity and immunoreactivity decreased to virtually undetectable levels after exposure of the cells to phorbol 12-myristate 13-acetate (PMA), associated with a commensurate increase in particulate kinase activity and immunoreactivity. In intact cells, PMA appeared to cause a shift of immunoreactive protein kinase C from the cytosol to the perinuclear region, as assessed by immunofluorescent microscopy; however; subcellular fractionation revealed that PMA caused increases in the protein kinase C activity associated primarily with non-nuclear membranes. Exposure of the cells to sn-1,2-dioctanoylglycerol resulted in a modest and transient membrane association of protein kinase C, whereas platelet-derived growth factor, fibroblast growth factor, and bombesin caused no detectable increases in the membrane association of the kinase. Activation of protein kinase C by growth factors in fibroblasts may occur without the gross disturbances in intracellular kinase location which occur in response to phorbol esters.

Insulin action in normal and protein kinase C-deficient rat hepatoma cells. Effects on protein phosphorylation, protein kinase activities, and ornithine decarboxylase activities and messenger ribonucleic acid levels.

Insulin and tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) share some biological activities in normal hepatocytes and in some lines of cultured hepatoma cells. To investigate the possibility that some of these common effects might involve a common pathway, we examined the effects of insulin and PMA on several biological processes in normal and protein kinase C-deficient H4IIE rat hepatoma cells. Protein kinase C deficiency was achieved by preincubating the cells in high concentrations of PMA, and was documented by direct enzyme measurement in soluble and particulate cellular fractions, and by analysis of immunoreactive protein kinase C concentrations in whole cellular homogenates. In the protein kinase C-deficient cells, the following actions of insulin remained at near normal levels: stimulated phosphorylation of the ribosomal protein S6; activation of a ribosomal S6 protein kinase; and increases in ornithine decarboxylase activity and mRNA accumulation. PMA stimulated all of these responses in the normal cells, but none of them in the PMA-pretreated cells. We conclude that insulin can exert some of its actions in a normal manner in protein kinase C-deficient H4IIE hepatoma cells (ATCC CRL 1548) and that some of the actions insulin holds in common with PMA may be due to common activation of one or more distal pathways. A candidate for such a distal step is activation of the ribosomal protein S6 protein kinase.

Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts.

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine fibroblasts after exposure to a variety of growth-promoting factors. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal growth factor, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for 16 h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent fibroblasts in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.

Deoxyadenosine inhibits DNA synthesis in cultured human fibroblasts.

The rate of DNA synthesis in cultured diploid fibroblasts, nonmalignant human cells, is decreased by 50 microM 2'-deoxyadenosine when adenosine deaminase is inhibited and 2'-deoxyadenosine is phosphorylated to dATP. No inhibiton of DNA synthesis occurs with 100 microM adenosine under identical conditions or with 50 microM deoxyadenosine when adenosine deaminase is not blocked. Inhibition of DNA synthesis may be an important link between adenosine deaminase deficiency and severe combined immunodeficiency if the tissue culture model is relevant to lymphocyte function in man.