Richard Balakir

Evidence of a direct role for Bcl-2 in the regulation of articular chondrocyte apoptosis under the conditions of serum withdrawal and retinoic acid treatment.

The regulation of chondrocyte apoptosis in articular cartilage may underlay age‐associated changes in cartilage and the development of osteoarthritis. Here we demonstrate the importance of Bcl‐2 in regulating articular chondrocyte apoptosis in response to both serum withdrawal and retinoic acid treatment. Both stimuli induced apoptosis of primary human articular chondrocytes and a rat chondrocyte cell line as evidenced by the formation of DNA ladders. Apoptosis was accompanied by decreased expression of aggrecan, a chondrocyte specific matrix protein. The expression of Bcl‐2 was downregulated by both agents based on Northern and Western analysis, while the level of Bax expression remained unchanged compared to control cells. The importance of Bcl‐2 in regulating chondrocyte apoptosis was confirmed by creating cell lines overexpressing sense and antisense Bcl‐2 mRNA. Multiple cell lines expressing antisense Bcl‐2 displayed increased apoptosis even in the presence of 10% serum as compared to wild‐type cells. In contrast, chondrocytes overexpressing Bcl‐2 were resistant to apoptosis induced by both serum withdrawal and retinoic acid treatment. Finally, the expression of Bcl‐2 did not block the decreased aggrecan expression in IRC cells treated with retinoic acid. We conclude that Bcl‐2 plays an important role in the maintenance of articular chondrocyte survival and that retinoic acid inhibits aggrecan expression independent of the apoptotic process. J. Cell. Biochem. 71:302–309, 1998.

Bcl‐2 regulates chondrocyte morphology and aggrecan gene expression independent of caspase activation and full apoptosis

Bcl‐2 is widely expressed in a variety of cell types and is known to block apoptosis through a conserved pathway. However, recent reports have demonstrated that Bcl‐2 regulates cell behavior independent of its control of apoptosis. Chondrocytes express a unique set of matrix proteins, including the proteoglycan aggrecan, and have been widely used to study the relationship between trophic factors and apoptosis. In this article, we report that Bcl‐2 affects the morphology and regulates the expression of aggrecan in a rat chondrocyte cell line (IRC). Endogenous Bcl‐2 and aggrecan mRNA were both down‐regulated in response to serum withdrawal in parental IRC cells, while constitutive expression of Bcl‐2 maintained aggrecan levels under conditions of serum withdrawal. In addition, expression of anti‐sense Bcl‐2 resulted in decreased aggrecan mRNA and produced a fibroblastic morphology compared with parental cells. The caspase inhibitor ZVAD‐fmk effectively blocked full apoptosis of IRC cells in response to serum withdrawal or anti‐sense Bcl‐2 but did not prevent the down‐regulation of aggrecan expression from either signal. These results suggest a novel role for Bcl‐2 in regulating the differentiated phenotype of chondrocytes and the expression of a differentiation‐specific gene independent of its control of apoptosis. J. Cell. Biochem. 74:576–586, 1999.

Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells

SummaryThein vivo andin vitro effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on calcium uptake by isolated chick duodenal cells were studied.In vivo, 1,25-(OH)2D3 given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion.In vitro, pre-incubation of 1,25-(OH)2D3 with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10−15m, was maximal at 10−13m, and was diminished at higher (10−11m) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D3 analogs was 1,25-(OH)2D3=1-(OH)D3>25-(OH)D3>1,24,25-(OH)3D3>24,25-(OH)2D3>D3. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)2D3 was five orders of magnitude greater than D3. Kinetically, 1,25-(OH)2D3 increased theVmax of calcium uptake; the affinity for calcium (Km=0.54mm) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)2D3,in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect of calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)2D3. These findings suggest that thein vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)2D3 induction of calcium transport and specific biochemical correlates.

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes

Abstract Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the 32P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of 32P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent Km for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10−8 m cAMP. The Ka values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.

Adenosine 3′:5′-cyclic phosphate-dependent and -independent protein kinases of renal brush border membranes: Solubilization, separation, and characterization of multiple forms

Abstract Multiple protein kinase activities were found in the luminal segment of the renal proximal tubule cell plasma membrane (brush border membrane). Membranes were extracted with Lubrol, with no loss in activity, and the extract was chromatographed on diethylaminoethyl cellulose with a salt gradient. With protamine as substrate, activity eluted in two peaks, designated I and IIb, and was cyclic AMP independent. With histone VII-S, one peak, designated IIa, appeared, which eluted slightly ahead of IIb and was cyclic AMP dependent. The three activities eluted in their original patterns following rechromatography. Histone kinase activity in the combined IIa+b fraction was stimulated threefold by cyclic nucleotides ( K a = 0.013 and 0.94 μM for cyclic AMP and cyclic GMP, respectively) by increasing V . Cyclic AMP binding activity eluted with histone kinase activity. Rechromatography of IIa+b on diethylaminoethyl cellulose containing 1 μ m cyclic AMP resulted in passage through the column of most of the histone kinase activity (IIa) prior to the salt gradient, but retention of kinase IIb, which again eluted in its original position. Characterization of the separated enzymes revealed that kinase I was highly specific for protamine and totally insensitive to cyclic AMP and a specific protein inhibitor of cyclic AMP-dependent kinases. Kinase IIa was relatively specific for histones and was completely inhibited by the protein inhibitor. Kinase IIb was nonspecific, catalyzing phosphorylation of protamine, casein, histones, and phosvitin in decreasing order of activity, and was insensitive to cyclic AMP and the protein inhibitor. Exposure of intact brush border membranes to elevated temperatures revealed that phosphorylation of intrinsic membrane proteins and protamine was thermolabile, whereas cyclic AMP-dependent histone kinase activity was relatively thermostable. These findings implicate cyclic AMP-independent protamine kinases in the cyclic AMP-independent autophosphorylation of the brush border membrane.

Characterization of dietary phosphorus-dependent duodenal calcium uptake in vitamin D-deficient chicks

The effect of dietary phosphorus on intestinal calcium uptake was examined in duodenal cells isolated from vitamin D-deficient chicks. Cells from chicks on a high phosphorus diet accumulated calcium at a rate 38% higher than cells from animals on a normal phosphorus diet. Diet high in calcium did not affect calcium absorption in duodenal cells. The dietary phosphorus effect on calcium absorption was specific. Uptake of α-methyl glucoside was not altered. Increase in calcium absorption by a high phosphorus diet was not due to a change in cellular energy metabolism nor to the content of phosphorus in cells. Kinetically, a high phosphorus diet increased the Vmax of calcium uptake; the affinity for calcium was unaffected. The effectiveness of dietary phosphorus to enhance the intestinal calcium uptake could also be demonstrated in brush border membrane vesicles. The increase in calcium uptake was not due to an alteration in membrane binding capacity nor to calcium efflux from vesicles. To test the hypothesis that a high phosphorus diet may affect membrane transport by altering phospholipid metabolism in duodenal cells, we examined the phospholipid content in isolated brush border membranes. The content of phosphatidylcholine, phosphatidylserine, phosphatidyinositol and phosphatidylethanolamine was not altered by the high phosphorus diet. These findings suggest that the vitamin D-independent and dietary phosphorus-dependent effect on intestinal calcium absorption was primarily due to a change in the calcium flux at the luminal side of the cells. However, the precise mechanism is still not clear.