The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C.

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.

Arachidonic acid directly mediates the rapid effects of 24,25-dihydroxyvitamin D3 via protein kinase C and indirectly through prostaglandin production in resting zone chondrocytes.

Prior studies have shown that 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)2D3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH)2D3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [3H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH)2D3 inhibited [3H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH)2D3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKCalpha; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKCalpha was inhibited, and matrix vesicle PKCzeta was stimulated; these effects were blocked by 24,25-(OH)2D3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE2, had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH)2D3. Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. Gamma-linolenic acid, an unsaturated precursor of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative regulator of 24,25-(OH)2D3 action in resting zone chondrocytes.

Monounsaturated fatty acid (avocado) rich diet for mild hypercholesterolemia.

To assess the effect of a high monounsaturated fatty acids (MFA) diet on serum lipids, 30 healthy adult normolipidemic volunteers and 37 adult patients with mild hypercholesterolemia (5.4-9.3 mmol/l), 15 of them also with hypertriglyceridemia (2.3-4.8 mmol/l), were studied. Fifteen healthy and 30 hypercholesterolemic subjects (15 of them with associated type 2 diabetes mellitus) received an avocado enriched diet (2000 Kcal, lipids 53% MFA 49 g saturated/unsaturated ratio 0.54), and seven non-diabetic hypercholesterolemic individuals received an isocaloric control diet (MFA 34 g, saturated/unsaturated ratio 0.7). Serum total cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride concentrations were measured before and after a 7-day diet period. In healthy individuals a 16% decrease of serum total cholesterol level followed the high MFA diet, while it rose after the control diet (p < 0.001 between diets). In hypercholesterolemic subjects a significant (p < 0.01) decrease of serum total cholesterol (17%), LDL-cholesterol (22%) and triglycerides (22%), and increase of HDL-cholesterol (11%) levels occurred with the avocado diet, while no significant changes were noticed with the control diet. High lipid, high MFA-avocado enriched diet can improve lipid profile in healthy and especially in mild hypercholesterolemic patients, even if hypertriglyceridemia (combined hyperlipidemia) is present.

Head and neck manifestations of the chronic graft vs. host disease.

Bone marrow transplantations are being used with increasing frequency in the treatment of patients with leukemia and aplastic anemia. The graft-vs.-host disease (GVHD) is a serious complication that affects long-term survivors following bone marrow transplantation. It is the result of an immunologic reaction mounted by the grafted reticuloendothelial cells against the tissues of the recipient, and it affects multiple organ systems. Involvement of the skin and mucosal surfaces of the head and neck region, in particular the oral cavity, occurs in a large number of patients with GVHD. In this report we present four patients with GVHD in whom mucosal lesions and infections of the head and neck region were prominent features. Our observations indicate that the clinical and histological characteristics of these lesions vary according to the time elapsed from the onset of the disease. Therefore, clinical examination of the head and neck region and biopsy of the oral mucosa are important not only in the diagnosis of the GVHD, but also in the evaluation of its progress and response to treatment.