Macrophages, multinucleated giant cells, and apoptosis in HIV+ patients and normal blood donors

Clearance of apoptotic debris is carried out by cells of the monocyte/macrophage lineage and, as other macrophage functions, it can be altered in AIDS, leading to the accumulation of apoptotic cells observed in this disease. In this study we evaluated the ability of macrophages from human immunodeficiency virus (HIV)-infected patients to differentiate and to clear apoptotic debris in prolonged in vitro cultures. Peripheral blood mononuclear cells (PBMC) from infected hemophilia patients were cultured in the absence of exogenously added stimulators and the organization and morphological characteristics of the cultures were analyzed and correlated with clinical staging of the patients. Cell aggregates of different sizes involving macrophages and lymphocytes were formed in cultures from asymptomatic HIV+ patients (CDC groups II-III) and controls and in 4/7 group IV C2 HIV+ patients. In order to obtain viable and organized cultures, cells had to be handled carefully, allowing contact and undisturbed sedimentation in round-bottom tubes. Multinucleated giant cells (MGC) were formed through macrophage fusion after 5 days of culture in HIV- controls, group II and III patients, and some of the group IV C2 patients, while scarce formation of MGC was observed in AIDS patients or patients with advanced HIV disease. This paucity was correlated with impaired dead cell removal and accumulation of apoptotic debris. Viability of macrophages and MGC was reduced after 15 days. MGC and the macrophages (either free or in cell aggregates) were able to remove dead cells, clearing the cultures of cell debris. Furthermore, in group II and III HIV+ hemophilic patients, increased macrophage-MGC phagocytic activity, suggesting in vivo activation, was frequently observed. In HIV+ patients with AIDS or advanced HIV disease (CDC groups IV A, IV C1, and IV D) dead cell removal was impaired and apoptotic debris accumulated. Long-term cultures of unstimulated PBMC are an interesting model for studying the role of macrophages and/or MGC in the removal of dead cells as well as examining the cellular milieu in which HIV replicates in an individual host.     

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.     

Effects of methotrexate on differentiation of monocytes and production of cytokine inhibitors by monocytes

OBJECTIVE: To examine the potential of methotrexate (MTX) to act as a differentiation-stimulating factor for monocytes, which could explain the antiinflammatory properties of this agent in the treatment of rheumatoid arthritis (RA). METHODS: Fluorescence-activated cell sorter analysis was used to measure the changes in antigen expression (CD11b/c, CD16, CD64, CD14, CD68, and CD95) in response to MTX, 1,25-OH-cholecalciferol (1,25-OH-CCF), and granulocyte-macrophage colony-stimulating factor in the human monoblastic leukemia cell line U937, bone marrow mononuclear cells (BMMC), and peripheral blood mononuclear cells (PBMC). Release of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor a, and soluble tumor necrosis factor receptors (sTNFR) p55 and p75 during the differentiation in vitro was assessed by immunoassay in the culture supernatants. RESULTS: MTX alone and in combination with 1,25-OH-CCF markedly stimulated the differentiation of the monocytic U937 cells and simultaneously increased Fas-antigen expression. Differentiation was associated with enhanced IL-1Ra and sTNFR p75 release from U937 cells. MTX had fewer effects on phenotypic differentiation of human BMMC and PBMC, but did stimulate IL-1Ra release and inhibit IL-1beta synthesis in BMMC. CONCLUSION: MTX acts as a strong differentiation factor for immature and undifferentiated monocytic cells. Differentiation in vitro is associated with an increase in natural cytokine inhibitor release and a simultaneous down-regulation of IL-1beta. These findings may explain the marked clinical antiinflammatory effects of MTX when used in the treatment of RA.     

Induction of surface tumor necrosis factor (TNF) expression and possible facilitation of surface TNF release from human monocytic cells by granulocyte-macrophage colony-stimulating factor or gamma interferon in combination with 1,25-dihydroxyvitamin D3

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can regulate monocyte maturation and activation. Using the human monocytoid cell line U937, we have shown that these agents increase surface tumor necrosis factor (TNF) expression without directly affecting TNF release. GM-CSF and IFN-gamma combined with 1,25(OH)2D3 increased cellular TNF secretion to levels not seen with these agents alone. Ability to express and secrete TNF in part depended on degree of monocytic maturation. The combination of 1,25(OH)2D3 and GM-CSF, however, facilitated lipopolysaccharide (LPS)-mediated release of surface TNF from U937 cells, an effect that was temporally independent of maximal maturation. 1,25(OH)2D3 plus IFN-gamma was less effective than 1,25(OH)2D3 plus GM-CSF at facilitating TNF secretion. We postulate that 1,25(OH)2D3 and GM-CSF are required together to prime a specific mechanism, probably a protease, which cleaves TNF from the surface of monocytic cells. This protease, once primed, can be activated by a secondary stimulus such as LPS.     

Increased CD4-positive monocytes in HIV-infected haemophilic patients

The monocyte-macrophage system is known to play a central role in HIV infection, and expression of CD4 on the surface of monocytes/macrophages is important, since this molecule is a key factor for the entrance of HIV into susceptible cells. In this paper we evaluated the expression of CD4 in monocytes of haemophilic patients (He) who had been infected with HIV (HIV + He) through transfusion of contaminated plasma concentrates. Thirty seropositive patients (HIV + He), 10 seronegative He patients (HIV-He) and 20 voluntary normal blood donors were studied. Phenotypic evaluation of monocytes was performed by flow cytometry of peripheral blood stained with anti-CD45, -CD3, -CD4 and -CD14 monoclonal antibodies. The percentage of CD4 monocytes was increased in all HIV+ patients groups, but it was highest in those belonging to Groups III and IV A of the CDC classification. Furthermore, the median of fluorescence intensity of CD4+ monocytes from individual patients was shifted to the right, indicating expression of increased numbers of CD4 molecules on the cell membrane of monocytes. This could in turn favour HIV infection and viral persistence, facilitating in vivo dissemination of the virus.     

In vivo parathyroid hormone stimulates in vitro bone resorption by bovine monocytes

Cells of the monocyte-macrophage lineage have been thought to play a role in bone resorption. We examined the effects of in vivo administration of parathyroid hormone and 1,25-dihydroxyvitamin D3 on the ability of monocytes to degrade bone in vitro. Administration of parathyroid hormone for 4 d resulted in sustained hypercalcemia and a transient 1-d increase in plasma 1,25-dihydroxyvitamin D3. Parathyroid hormone significantly stimulated bone degradation by monocytes 2.6 times more than that of pretreatment controls. Parathyroid hormone treatment significantly enhanced (threefold) release of superoxide anion by monocytes stimulated with phorbol 12-myristate 13-acetate and increased migration of monocytes to bone particles in vitro. Continuous 7-d infusion of 1,25-dihydroxyvitamin D3 (50 micrograms/d) elevated plasma 1,25-dihydroxyvitamin D3 until infusions were discontinued. Increased 1,25-dihydroxyvitamin D3 was associated with hypercalcemia, which continued for several days postinfusion. In vivo administration of 1,25-dihydroxyvitamin D3 did not affect in vitro ability of monocytes to degrade bone. We concluded that in vivo administration of parathyroid hormone enhanced in vitro responsiveness of isolated monocytes in a manner consistent with a role for monocytes in bone remodeling. Furthermore, these data suggested that circulating monocytes could be a useful experimental model for further studies on parathyroid hormone responsiveness and bone resorption for the cow with milk fever.     

Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis

The serum level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D], the biologically most potent metabolite of vitamin D, is tightly regulated within narrow limits in human healthy adults. 1,25-(OH)2D deficiency is rare and is associated with disturbances in calcium and bone metabolism. We have previously reported a marked decrease in serum levels of 1,25-(OH)2D in human immunodeficiency virus (HIV)-infected patients. The present study was designed to further examine the causes and consequences of severe 1,25-(OH)2D deficiency in these patients. The design was a prospective cohort study. Fifty-four HIV-infected patients clinically classified according to the revised criteria from Centers for Disease Control and Prevention and healthy controls were studied. Parameters related to vitamin D and calcium metabolism as well as immunological and nutritional status were determined. Twenty-nine of the patients (54%) had serum levels of 1,25-(OH)2D below the lower reference limit, and 18 of these had undetectable levels. In contrast, HIV-infected patients had normal serum levels of 25-hydroxyvitamin D and vitamin D-binding protein. HIV-infected patients as a group had modestly depressed serum calcium and PTH levels. There were, however, no correlations between these parameters and serum levels of 1,25-(OH)2D. There were no differences in serum calcium or PTH levels or nutritional status when patients with severe 1,25-(OH)2D deficiency were compared to other patients, but patients with undetectable 1,25-(OH)2D had significantly elevated serum phosphate levels. Furthermore, patients with undetectable 1,25-(OH)2D levels were characterized by advanced clinical HIV infection, low CD4+ lymphocyte counts, and high serum levels of tumor necrosis factor-alpha (TNFalpha). We conclude that inadequate 1alpha-hydroxylation of 25-hydroxyvitamin D seems to be the most likely cause of 1,25-(OH)2D deficiency in HIV-infected patients, possibly induced by an inhibitory effect of TNFalpha. The low 1,25-(OH)2D and high TNFalpha levels observed may impair the immune response in HIV-infected patients both independently and in combination and may represent an important feature of the pathogenesis of HIV-related immunodeficiency. Markedly depressed 1,25-(OH)2D serum levels are also present in certain other disorders characterized by immunological hyperactivity. Thus, the findings in the present study may not only represent a previously unrecognized immune-mediated mechanism for induction of 1,25-(OH)2D deficiency in human disease, but may also reflect the importance of adequate serum levels of 1,25-(OH)2D for satisfactory performance of the immune system in man.     

Expression of the receptor for urokinase-type plasminogen activator in normal and neoplastic blood cells and hematopoietic tissue

Expression of the receptor for the urokinase type plasminogen activator (uPAR) has been studied by flow cytometry and immunohistology in normal blood and bone marrow cells, in vitro activated lymphoid cells, and tissue samples from reactive lymph nodes (n = 6), thymus (n = 2) and malignant lymphomas (n = 82), or leukemias (n = 32). HL-60 myeloid precursor cells and CD34-positive normal stem cells also were analyzed. In the normal cells, staining was confined to monocytes, macrophages, neutrophils, and myeloid precursors. No labelling was seen of normal or activated lymphoid cells. Purified CD34-positive hematopoietic progenitors were uPAR negative, but expressed uPAR during differentiation in short-term liquid culture stimulated in vitro by recombinant interleukin (IL)-1, IL-3, IL-6, granulocyte-macrophage colony stimulating factor (CSF), granulocyte-CSF, and stem cell factor. Enhanced uPAR expression was also seen in HL-60 cells after induction of differentiation with dimethyl sulfoxide or 1 alpha,25-dihydroxyvitamin D3. In lymphomas and leukemias, the staining pattern was similar to that seen in the normal cells with labelling of monocytic and myeloid that seen in the normal cells with labelling of monocytic and myeloid malignancies, but not of the neoplastic cells in B-cell or T-cell lymphomas or Hodgkin's disease. In conclusion, uPAR is a differentiation marker for myeloid and monocytic cells, and may act to facilitate migration of these cells in normal and pathologic conditions by cell-associated plasminogen activation. Whether expression of uPAR in myeloid and monocytic malignancies relates to their growth and behavior will be an important topic for investigations in the future.     

Lung cancer in patients with HIV-infection

PURPOSE: To identify and review the clinical characteristics and natural history of lung cancer in HIV-seropositive patients. A secondary objective was to compare the clinical features of HIV-seropositive and HIV-indeterminate lung cancer cases at our institution. PATIENTS AND METHODS: Sixteen patients with HIV infection and lung cancer were diagnosed between January 1988 and March 1995 at our institution and the clinical records were reviewed. HIV-indeterminate lung cancer cases were identified by the Albany Medical Center Hospital (AMCH) Tumor Registry. A Medline database search of HIV infection/AIDS and lung cancer was undertaken through December 1994. The New York State Department of Health (NYSDOH), Bureau of Cancer Epidemiology provided information on the incidence of lung cancer among residents of New York State by county of residence. Case reports and series regarding the clinical features of HIV-seropositive patients with lung cancer were reviewed. A more focused comparison between HIV-seropositive and HIV-indeterminate male lung cancer cases between 35 and 54 years of age at our institution was performed. The following clinical variables were identified in our 16 patients and 109 cases extracted from available clinical reports: sex, age, year and county of residence at time of lung cancer diagnosis, cigarette smoking history, HIV risk behavior, CD4 count at time of lung cancer diagnosis, CDC classification of HIV disease, interval in months from time of HIV seropositivity to lung cancer diagnosis, pathology and stage of lung cancer, performance status, treatment, response, and survival. RESULTS: Lung cancer in HIV-seropositive patients is characterized by the following: a younger age at time of diagnosis when compared to HIV-indeterminate cases; the majority of cases occur in a background of extensive cigarette smoking; over 80% of patients present with advanced stage of lung cancer (stage III and IV); up to 50% of cases have asymptomatic to mildly symptomatic HIV infection with a median CD4 lymphocyte count of 233 per microliter; there is a predominance of adenocarcinoma histopathology; and shortened survival when compared to HIV-indeterminate cases. CONCLUSION: Current reports of lung carcinoma in HIV-seropositive patients suggest that the natural history of this disease is different than in HIV-indeterminate cases. Lung cancer must be considered in the differential diagnosis of a solitary mass lesion on chest X-ray in HIV-seropositive patients.     

Tandem-in-time mass spectrometry method for the sub-parts-per-trillion determination of 2,3,7,8-chlorine-substituted dibenzo-p-dioxins and -furans in high-fat foods

Phosphorylation of the nucleosome core histone H3 (H3) on Ser-10 is thought to be a prerequisite for chromatin condensation at mitosis. Although during interphase, cell differentiation, or mitogenic activation of quiescent cells, changes in chromatin structure that involve local chromatin condensation/decondensation also occur, little is known about H3 phosphorylation during these transitions. Using the recently developed sensitive marker to monitor H3 phosphorylation, namely, the mAb that recognizes the phosphorylated epitope of H3 (anti-H3-P mAb), the status of H3 phosphorylation was assayed in individual human lymphocytes after their mitogenic stimulation (G0 to G1 transition) and in human leukemic HL-60 cells induced to differentiate by all-trans-retinoic acid (RA), 1,25-dihydroxyvitamin D3 (vit D3), dimethyl sulfoxide (DMSO), or phorbol myristate acetate (PMA). Multiparameter flow cytometry was used to correlate H3 phosphorylation with cell cycle position. The specificity of the anti-H3-P mAb was confirmed by the loss of its binding following cell treatment with alkaline phosphatase. The presence of phosphorylated H3 was detected during interphase in HL-60 cells and in normal lymphocytes at a level severalfold lower than during mitosis. No significant changes in H3 phosphorylation were observed during lymphocyte stimulation. Unexpectedly, the level of H3 phosphorylation was over fourfold higher in monocytes than in lymphocytes or granulocytes from peripheral blood. The punctate pattern of labeling with anti-H3-P mAb in monocyte nuclei suggests that H3 is phosphorylated in small clusters of adjacent nucleosomes. Differentiation of HL-60 cells was accompanied by a rise in H3 phosphorylation, which was higher after induction by RA, vit D3, and PMA (approx. threefold) than after DMSO (approximately 20%). The data indicate that in addition to being a critical event during chromatin condensation at mitosis, H3 phosphorylation plays a role during chromatin changes accompanying differentiation of HL-60 cells, in particular, along the monocytic lineage. The high level of H3 phosphorylation in monocytes may serve as a marker of these cells and is being explored as a possible diagnostic and prognostic tool in monocytic leukemias.     

Ethical concerns in modern medical practice

Differentiation of HL-60 cells along the granulocytic lineage by DMSO in the presence of transforming growth factor-beta and low concentrations of 1,25-dihydroxyvitamin D3 leads to the upregulation of 5-lipoxygenase activity in 100,000 g supernatants and intact cells to levels which are comparable to normal granulocytes. Similarly, differentiation of the human monocytic cell line Mono Mac 6 by 1,25-dihydroxyvitamin D3 and transforming growth factor-beta strongly upregulates the 5-lipoxygenase pathway. Here, we describe an assay system for leukotriene biosynthesis inhibitors which is based on the in-vitro differentiation of HL-60 and Mono Mac 6 cells. Different leukotriene biosynthesis inhibitors like the nonredox type inhibitor ZM 230487, the redox type inhibitor BW A4C and the FLAP inhibitor MK886 were tested and the results were compared with an assay system based on normal human granulocytes. ZM 230487, BWA4C and MK886 showed similar potencies in these cell lines as compared to normal leukocytes. Thus, the in-vitro differentiation of HL-60 and Mono Mac 6 cells provides an excellent model for the screening of drugs affecting the 5-lipoxygenase pathway.     

A group of deltanoids (vitamin D analogs) regulate cell growth and proliferation in small cell carcinoma cell lines

A group of deltanoids has been used for studying the inhibition of cell growth and proliferation in two small cell lung carcinoma (SCLC) in vitro. The biologically active deltanoid, 1,25 dihydroxyvitamin D3 (1,25 (OH)2D3), has functions beyond its classical roles of stimulating calcium transport and serum calcium. It also causes the differentiation of a variety of precursor cells and suppresses growth. Although 1,25(OH)2D3 has an inhibitory effect on growth of certain malignant cells, its hypercalcemic effect has prevented clinical applications. Several new deltanoids, which showed comparable or even greater abilities to induce differentiation and to inhibit proliferation, have been identified. Furthermore, these synthetic deltanoids have been shown to be less effective on calcium metabolism and less hypercalcemic. We have selected four synthetic deltanoids; MC-903, 1 alpha-OH-pregnacalciferol, 19-nor-24 homo, and 19-nor-22(E). When compared with 1,25 (OH)2D3, these deltanoids showed considerable potency on cell growth and proliferation in the NCI-H82 and the NCI-H209 SCLC lines. Cells were treated with various concentrations of deltanoids. They inhibited the growth and proliferation of both SCLC cells in vitro in a time-and dose-dependent manner, as determined by cell number and 3H-thymidine uptake. 19-nor-22(E) showed an antiproliferative effect significantly comparable to 1,25(OH)2D3 in the NCI-H82 cell line 1 alpha-OH-pregnacalciferol, 19-nor-24 homo, and 19-nor-22(E) inhibited the cell growth in the NCI-H209 cells within the same significance as 1,25 (OH)2D3. The degree of the suppressive effect of the deltanoids was cell line dependent.     

EB 1089, a novel vitamin D analogue, has strong antiproliferative and differentiation inducing effects on cancer cells

EB 1089 is a novel vitamin D analogue which in vitro strongly inhibits the proliferation of U937 histiocytic lymphoma cells and MCF-7 breast cancer cells, with a potency of 50 to 100 times that of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Studies of c-myc and c-fos expression in MCF-7 cells and of differentiation markers in U937 cells show that growth inhibition by EB 1089 is accompanied by induction of differentiation. The ability of EB 1089 to affect calcium metabolism in vivo in rats is decreased, compared to 1,25(OH)2D3. This low calcemic effect combined with the strong biological effect on cancer cells in vitro, makes EB 1089 an interesting candidate for treatment of cancer.     

A new method to isolate large numbers of rabbit osteoclasts and osteoclast-like cells: application to the characterization of serum response element binding proteins during osteoclast differentiation

We have developed a new method that allows the purification of large numbers of both authentic osteoclasts (OCs) and in vitro differentiated osteoclast-like cells (OCLs) from rabbits. We characterized the OCLs in terms of the expression of different phenotypic markers of OC differentiation and their ability to resorb bone. The method provides a system for performing biochemical and molecular studies of OC differentiation and function in a single species. We used this system to characterize the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of proteins that bind to the serum response element (SRE) of the c-fos promoter. We found that OCLs and OCs displayed similar SRE-binding activities, including the serum response factor (SRF). This pattern is established in a time-dependent and cell-specific manner in response to long-term treatment of rabbit bone marrow by 1,25(OH)2D3. Thus, 1,25(OH)2D3 can modulate SRF and/or SRF-related protein. This finding may contribute to understanding the role of c-Fos in the regulation of OC differentiation.     

The synergistic effects of vitamin D metabolites and transforming growth factor-beta on costochondral chondrocytes are mediated by increases in protein kinase C activity involving two separate pathways

Transforming growth factor-beta (TGFbeta), as well as the vitamin D3 metabolites 1,25-dihydroxyvitamin D3 (1,25) and 24,25-dihydroxyvitamin D3 (24,25), regulate chondrocyte differentiation and maturation during endochondral bone formation. Both the growth factor and secosteroids also affect protein kinase C (PKC) activity, although each has its own unique time course of enzyme activation. Vitamin D3 metabolite effects are detected soon after addition to the media, whereas TGFbeta effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGFbeta on chondrocyte differentiation, matrix production, and proliferation. We also examined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is mediated by PKC. This study used a chondrocyte culture model developed in our laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less mature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGFbeta showed synergistic effects on RC alkaline phosphatase-specific activity (ALPase). No similar effect was found when 24,25 plus TGFbeta was added to GC cells or when 1,25 plus TGFbeta were added to GC or RC cells. The addition of 1,25 plus TGFbeta and 24,25 plus TGFbeta to GC and RC cells, respectively, produced a synergistic increase in [35S]sulfate incorporation and had an additive effect on [3H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGFbeta on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGFbeta for 12 h produced a synergistic increase in PKC activity. Moreover, a similar effect was found when 24,25 was added for only the last 90 min of a 12-h incubation. However, a synergistic effect could not be found when 24,25 was added for the last 9 min or the first 90 min of incubation. To further understand how 24,25 and TGFbeta may mediate the observed synergistic increase in PKC activity, the pathways potentially leading to activation of PKC were examined. It was found that 24,25 affects PKC activity through production of diacylglycerol, not through activation of G protein, whereas TGFbeta only affected PKC activity through G protein. The results of the present study indicate that vitamin D metabolites and TGFbeta produced a synergistic effect that is maturation-dependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGFbeta was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGFbeta through G protein activation.