Leonard Rifas

The Mechanism of Phosphorus as a Cardiovascular Risk Factor in CKD

Hyperphosphatemia and vascular calcification have emerged as cardiovascular risk factors among those with chronic kidney disease. This study examined the mechanism by which phosphorous stimulates vascular calcification, as well as how controlling hyperphosphatemia affects established calcification. In primary cultures of vascular smooth muscle cells derived from atherosclerotic human aortas, activation of osteoblastic events, including increased expression of bone morphogenetic protein 2 (BMP-2) and the transcription factor RUNX2, which normally play roles in skeletal morphogenesis, was observed. These changes, however, did not lead to matrix mineralization until the phosphorus concentration of the media was increased; phosphorus stimulated expression of osterix, a second critical osteoblast transcription factor. Knockdown of osterix with small interference RNA (siRNA) or antagonism of BMP-2 with noggin prevented matrix mineralization in vitro. Similarly, vascular BMP-2 and RUNX2 were upregulated in atherosclerotic mice, but significant mineralization occurred only after the induction of renal dysfunction, which led to hyperphosphatemia and increased aortic expression of osterix. Administration of oral phosphate binders or intraperitoneal BMP-7 decreased expression of osterix and aortic mineralization. It is concluded that, in chronic kidney disease, hyperphosphatemia stimulates an osteoblastic transcriptional program in the vasculature, which is mediated by osterix activation in cells of the vascular tunica media and neointima.

T cell activation induces human osteoclast formation via receptor activator of nuclear factor κB ligand-dependent and -independent mechanisms

In unstimulated conditions, osteoclast (OC) formation is regulated by stromal cell production of the key osteoclastogenic factors receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). However, the mechanisms of accelerated osteoclastogenesis and bone loss characteristic of inflammatory conditions are poorly understood but appear to involve T cells. In addition, the mechanism by which OCs arise spontaneously in cultures of peripheral blood mononuclear cells in the absence of stromal cells or added cytokines remains unclear. Using a stromal cell free human osteoclast generating system, we investigated the ability of activated T cells to support osteoclastogenesis. We show that when activated by phytohemagglutinin‐P (PHA), T cells (both CD4+ and CD8+) stimulate human OC formation in vitro. Although both soluble M‐CSF and RANKL were detected in activated T cell supernatants, the presence of M‐CSF was not essential for macrophage survival or RANKL‐dependent osteoclast formation, suggesting that other soluble T cell‐derived factors were capable of substituting for this cytokine. We also found that saturating concentrations of osteoprotegerin (OPG) failed to neutralize 30% of the observed OC formation and that T cell conditioned medium (CM) could superinduce osteoclastogenesis in cultures of purified monocytes maximally stimulated by RANKL and M‐CSF. Together, these data suggest that activated T cells support osteoclastogenesis via RANKL‐dependent and ‐independent mechanisms. Although not relevant for T cell‐induced osteoclastogenesis, secretion of soluble M‐CSF is a previously undescribed property of activated T cells.

Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts

Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up‐regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up‐regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up‐regulated by activated T cell conditioned medium, its inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF‐related apoptosis‐inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up‐regulation of bone turnover via coupling. J. Cell. Biochem. 88: 650–659, 2003.

A novel T cell cytokine, secreted osteoclastogenic factor of activated T cells, induces osteoclast formation in a RANKL-independent manner.

OBJECTIVE Chronic T cell activation is central to the etiology of rheumatoid arthritis (RA), an inflammatory autoimmune disease that leads to severe focal bone erosions and generalized systemic osteoporosis. Previous studies have shown novel cytokine-like activities in medium containing activated T cells, characterized by potent induction of the osteoblastic production of interleukin-6 (IL-6), an inflammatory cytokine and stimulator of osteoclastogenesis, as well as induction of an activity that directly stimulates osteoclast formation in a manner independent of the key osteoclastogenic cytokine RANKL. This study was undertaken to identify the factors secreted by T cells that are responsible for these activities. METHODS Human T cells were activated using anti-human CD3 and anti-human CD28 antibodies for 72 hours in AIM V serum-free medium to obtain T cell-conditioned medium, followed by concentration and fractionation of the medium by fast-protein liquid chromatography. Biologically active fractions were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major bands were analyzed by mass spectrometry, and a major candidate protein was identified. This novel cytokine was cloned, and its expression was analyzed using recombinant DNA technologies. RESULTS A single novel cytokine that could induce both osteoblastic IL-6 production and functional osteoclast formation in the absence of osteoblasts or RANKL and that was insensitive to the effects of the RANKL inhibitor osteoprotegerin was identified in the activated T cell-conditioned medium; this cytokine was designated secreted osteoclastogenic factor of activated T cells (SOFAT). Further analysis of SOFAT revealed that it was derived from an unusual messenger RNA splice variant coded by the threonine synthase-like 2 gene homolog, which is a conserved gene remnant coding for threonine synthase, an enzyme that functions only in microorganisms and plants. CONCLUSION SOFAT may act to exacerbate inflammation and/or bone turnover under inflammatory conditions such as RA or periodontitis and in conditions of estrogen deficiency.

A Novel T Cell Cytokine Stimulates Interleukin‐6 in Human Osteoblastic Cells

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a heavy lymphocytic infiltration into the synovial cavity, resulting in the secretion of a variety of cytokines which ultimately leads to destruction of joint tissue. Among the infiltrating cells are activated T cells which produce specific cytokines capable of osteoclast progenitor cell expansion, fusion, and activation. Cultures of activated human T cells and human osteoblasts (hOBs) were used to study the possibility that lymphokines may act on osteoblasts to produce the osteoclastogenic factor interleukin‐6 (IL‐6). Purified T cells were activated with a combination of anti‐CD3 and anti‐CD28 antibodies, cocultured with hOBs in direct physical contact or separated by a transwell system, and conditioned media (CM) were assayed for IL‐6 production. After a 72 h incubation period, activated T cell–hOB interaction resulted in a 100‐fold increase of IL‐6 production over basal levels. The immunosuppressant cyclosporine A (CsA) inhibited T cell tumor necrosis factor alpha and IL‐6 production but did not inhibit the T cell induction of IL‐6 from hOB. Assay of activated T‐cell CM on hOB revealed that a soluble factor, not cell‐cell contact, was the major inducer of IL‐6. The induction of IL‐6 mRNA by both activated T cell CM and CsA‐treated activated T cell CM was confirmed by Northern blot analysis. Neutralizing antibodies to IL‐13 and IL‐17 did not affect IL‐6 production. These findings suggest that activated T cells produce a novel, potent, IL‐6 inducing factor that may be responsible for the bone loss observed in RA patients.

The role of noggin in human mesenchymal stem cell differentiation

Noggin is a secreted protein that inhibits the binding of bone morphogenetic proteins (BMPs) to their cognate receptor. Its role in human mesenchymal stem cell differentiation has not been well studied. Here, we studied the effect of noggin on human mesenchymal stem cell differentiation induced by inflammatory cytokines (activated T‐cell conditioned medium (ACTTCM) or the combination of four T‐cell cytokines, TNF‐α, TGF‐β, IFN‐γ, and IL‐17 (TTII)), BMPs, or dexamthasone (DEX). HMSC treated with TTII alone rapidly induced alkaline phosphatase (AlkP) activity. Inclusion of noggin resulted in an additive effect. Noggin acted additively with DEX to induce a significantly higher level of AlkP induction than either noggin or DEX alone. Noggin was examined for its ability to inhibit mineralization in long‐term cultures of HMSC stimulated with BMP‐2, BMP‐6, BMP‐7, DEX, or TTII. Surprisingly, noggin alone induced mineralization while it did not inhibit mineralization induced by TTII or BMP‐2, BMP‐6, or BMP‐7. Interestingly, when HMSC were treated with both noggin and DEX they acted synergistically to induce mineralization nearly 3‐fold over DEX alone and 30‐fold over noggin alone. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis showed that T‐cell cytokines induced noggin, Runx2, BMP‐2, and osteocalcin gene expression, while noggin alone induced BMP‐2 and osteocalcin gene expression, but not Runx2, although it increased the expression of ActRII, a receptor for BMP‐2. These results suggest that in HMSC, the anabolic action of inflammation on bone formation occurs through the induction of noggin, which then induces BMP‐2 receptor and BMP‐2 leading to the activation of the differentiation process. J. Cell. Biochem. 100: 824–834, 2007.

SKELETAL CASEIN KINASE ACTIVITY DEFECT IN THE HYP MOUSE

Abstract: The Hyp mouse, a model for human X-linked hypophosphatemia (XLH), is characterized by phosphate wasting and defective mineralization. Since osteopontin (OPN) is considered pivotal for biological mineralization, we examined the biosynthesis of OPN in osteoblasts of +/Y and Hyp/Y mice. Immunoprecipitation analyses using a specific antibody to OPN revealed that Hyp/Y and +/Y osteoblasts secrete similar levels of OPN as determined by [35S]-methionine biosynthetic labeling, but a reduced phosphorylation was noted after 32P-PO4 biosynthetic labeling. Northern blot hybridization analysis of +/Y and Hyp/Y mice osteoblast mRNAs, using a cDNA probe for mouse OPN, revealed no difference in the steady state levels of osteopontin mRNA. Analysis of casein kinase II activity in +/Y and Hyp/Y mice osteoblast, kidney, heart and liver membrane fractions revealed that casein kinase II activity in the Hyp/Y mice osteoblasts and kidney is only 35%-50%, respectively, of that of the +/Y mice tissues. The accumulated data are consistent with a post-translation defect in the Hyp/Y mouse osteoblast which results in the under-phosphorylation of osteopontin and subsequent under-mineralization of bone matrix.

Altered Osteoblast Gluconeogenesis in X-Linked Hypophosphatemic Mice Is Associated with a Depressed Intracellular pH

We have studied gluconeogenesis and intracellular pH levels in normal (+/Y) and X-linked hypophosphatemic (Hyp/Y) mice. Compared with +/Y littermates, Hyp/Y mouse osteoblasts showed a higher rate of glucose production from fructose (10-fold), glutamine, and malate, but no significant difference when α-ketoglutarate was used as substrate. The activities of the pentose cycle enzymes, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase, were not different in the two osteoblast preparations. Examination of intracellular pH (pHi) using the double excitation of the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) revealed a significantly lower pHi in Hyp/Y mouse osteoblasts compared with +/Y mouse osteoblasts (7.01±0.03 n=10 versus 7.15±0.04 n=8, respectively; P<0.05). These results show for the first time that osteoblasts are capable of glucose production and that glucose production is altered in the Hyp/Y mouse osteoblast. As altered gluconeogenesis has been associated with reduced intracellular pH in other systems, a similar mechanism may be operative in the Hyp/Y mouse osteoblast. The observed defects may be intrinsic to the Hyp phenotype as the alterations in intracellular pH and gluconeogenesis persisted in vitro, or they may represent impressed memory from the in vivo state and the presumed circulating factor that influences phosphate transport.

Phosphate transport in osteoblasts from normal and X-linked hypophosphatemic mice.

Human hypophosphatemic vitamin D-resistant rickets (X-linked hypophosphatemia-XLH) is characterized by hypophosphatemia, a decreased tubular reabsorption of phosphate (Pi) and defective skeleton mineralization. Utilizing a mouse model (Hyp) of XLH, which demonstrates biological abnormalities and skeletal defects of XLH, we analyzed sodium-dependent phosphate transport in isolated osteoblasts derived from the calvaria of normophosphatemic and hypophosphatemic mice. Initial rates of phosphate uptake by normal and Hyp osteoblasts showed similar slopes. Osteoblasts from both normal and Hyp mice exhibited saturable, sodium-dependent phosphate transport with apparent Vmax and Km values not significantly different (normal mice, Vmax=24.30±3.45 nmol/mg prot. 10 min, Km=349.49±95.20 μmol/liter; Hyp mice, Vmax=23.03±3.41 nmol/mg prot. 10 min, Km=453.64±106.93 μmol/liter, n=24). No differences were found in the ability of normal and Hyp osteoblasts to respond to Pi transport after 5 hours of Pi deprivation. Both cell types exhibited a similar increase in cAMP in response to PTH. The accumulated results demonstrate that Pi uptake and transport in normal and Hyp mouse osteoblasts is a sodium-dependent saturable process. As osteoblast Pi uptake and transport is apparently normal in the Hyp mouse model of XLH, the “osteoblastic failure” described for the Hyp mouse should be attributed to other mechanism(s).

Monokines produced by macrophages stimulate the growth of osteoblasts.

We have previously reported that the J774A.1 macrophage-like tumor cell line produces two potent monokines which stimulate the growth of osteoblasts and chondrocytes. These growth factors, which have an affinity for heparin-agarose, have been termed HEP I (a 30 Kd PDGF-like molecule) and HEP II (an approximately 20 Kd molecule), respectively, based on their elution profile. Unlike HEP I, HEP II does not stimulate the growth of fibroblasts. Extensive biological and chromatographic studies disclosed that HEP II appears to be a unique bone cell mitogen unlike any known growth factor, including the FGFs, IL-1s, and TNFs, EGF, IGF-I and -II, TGF-beta, beta 2 microglobulin, G-CSF, CSF-1 and GM-CSF. To characterize more fully the effects of the macrophage-derived monokines on osteoblast growth and function, clones were derived from calvaria explant cultures. Two clones, SDFRC-2.05 and SDFRC-3, were developed and found to exhibit osteoblastic characteristics, including high levels of alkaline phosphatase, synthesis of type I but not type III collagen, and an increased intracellular cAMP production in response to PTH. The SDFRC-3 cells exhibited a polygonal morphology like that of the explant-derived cells while SDFRC-2.05 cells exhibited a more fibroblastic morphology. When tested on the explant cultures and clones, HEP I and HEP II were found to stimulate DNA synthesis and increase protein per culture, but decreased alkaline phosphatase activity. Clone SDFRC-3 was found to be more responsive to HEP II than clone SDFRC-2.05. Both monokines were found to be more potent mitogens for bone cells than TGF-beta. HEP II, but not HEP I or TGF-beta, induced a transformation of bone cells from a polygonal to a fibroblastic morphology, suggesting the induction of migration prior to proliferation. Thus, macrophages may be responsible not only for bone repair but also for ensuring the linkage of bone formation to resorption during physiological remodeling.