Ann E. Carlson

Dietary omega-3 fatty acids decrease mortality and Kupffer cell prostaglandin E2 production in a rat model of chronic sepsis.

We tested the hypothesis that substitution of omega-3 fat for dietary omega-6 fat would reduce mortality and decrease Kupffer cell prostaglandin E2 (PGE2) production in a rat model of chronic sepsis. Rats were fed via gastrostomy for 12 days with isonitrogenous, isocaloric diets containing 15% of calories as either safflower oil (omega-6) or a 10:1 mixture of menhaden oil (omega-3) and safflower oil. After five days of feeding, animals received an intra-abdominal abscess of defined bacterial content. Survivors were killed on post-laparotomy day 6 in conjunction with liver perfusion and protease liver digestion for Kupffer cell isolation. Kupffer cell PGE2 production was measured by radioimmunoassay after 18 hours of cell culture and again after stimulation with 0 LPS, 10 ng/ml LPS, and 10 micrograms/LPS. Mortality was decreased in menhaden oil-fed animals compared with safflower oil-fed animals (16% vs. 35%). Kupffer cell PGE2 production was decreased in menhaden oil-fed animals at 18 hours (354 +/- 54 vs. 570 +/- 95 pg/0.1 ml; p = 0.09) and after stimulation with 10 micrograms/ml LPS (140 +/- 41 vs. 288 +/- 45 pg/0.1 ml; p = 0.03) compared with safflower oil-fed animals.

Enhanced Osteoclastogenesis Causes Osteopenia in Twisted Gastrulation-Deficient Mice Through Increased BMP Signaling†‡

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1‐deficient (Twsg1−/−) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1−/− mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1−/− mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL‐mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1−/− mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1−/− mice was reversed in vitro in a dose‐dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.

Bone morphogenic protein 2 directly enhances differentiation of murine osteoclast precursors.

Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF‐κB ligand (RANKL) to enhance in vitro differentiation of osteoclast‐like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow‐derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p‐SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p‐SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL‐stimulated formation of multinuclear TRAP‐positive cells. The BMP antagonist noggin inhibited RANKL‐mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts‐like cells. J. Cell. Biochem. 109: 672–682, 2010.

Tenofovir treatment of primary osteoblasts alters gene expression profiles: Implications for bone mineral density loss

There is strong clinical evidence that implicates tenofovir in the loss of bone mineral density during treatment of human immunodeficiency virus infection. In this study, we sought to test the hypothesis that tenofovir treatment of osteoblasts causes changes in the gene expression profile that would impact osteoblast function during bone formation. Primary osteoblasts were isolated and then treated with the tenofovir prodrug, tenofovir disoproxil fumarate (TDF). Total RNA from TDF-treated and untreated osteoblasts were extracted and used for microarray analysis to assess TDF-associated changes in the gene expression profile. Strikingly, the changes in gene expression profiles involved in cell signaling, cell cycle and amino acid metabolism, which would likely impact osteoblast function in bone formation. Our findings demonstrate for the first time that tenofovir treatment of primary osteoblasts results in gene expression changes that implicate loss of osteoblast function in tenofovir-associated bone mineral density loss.

Role of matrix Gla protein in parathyroid hormone inhibition of osteoblast mineralization.

Parathyroid hormone (PTH) exerts biphasic effects on bone, dependent on the frequency and dose of administration. The catabolic actions of PTH on bone have been associated with continuous treatment, an increase in osteoblast-mediated resorption of bone via osteoclast activation, and inhibition of osteoblast activity and mineralization. Downregulation of differentiation markers and inhibition of mineralization by PTH have been reported in primary calvarial explants and osteoblast cell lines. Using MC3T3-E1 osteoblast-like cells, we have shown that matrix Gla protein (MGP) can be induced by PTH, and that this induction may explain the PTH-mediated inhibition of osteoblast biomineralization. MGP is a known inhibitor of mineralization, and mice deficient in Mgp show severe vascular calcification and premature bone mineralization. This review discusses the role of MGP in mineralization, comparing bone and vascular mineralization. In addition to MGP, the regulation and possible role of osteopontin, another known regulator of osteoblast mineralization, in PTH-mediated regulation of bone and vascular mineralization is discussed.

Tumor necrosis factor production by Kupffer cells requires protein kinase C activation

Tumor necrosis factor (TNF) has been proposed as a primary inflammatory mediator of septic shock. In vitro and in vivo studies indicate that endotoxin- or lipopolysaccharide (LPS)-activated macrophages are a principle source of TNF; however, membrane signal transduction and intracellular pathways by which LPS triggers TNF production in macrophages are unclear. Recent evidence indicates that specific protein phosphorylation via activation of protein kinase C (PKC) is an early, critical step in the signaling of macrophage TNF production by phorbol esters. We hypothesize that PKC activation is also required in LPS-signaled Kupffer cell (KC) TNF production. Murine KCs were obtained by liver perfusion and digestion and then stimulated with LPS (Escherichia coli O111:B4) or LPS in the presence of H-7, a selective PKC inhibitor. Conditioned media was collected at 3 hr for assay of TNF utilizing the L929 cytolysis bioassay standardized to murine-rTNF-alpha. We found that H-7 inhibited significantly LPS signaled TNF release at a concentration of 10 microM, while H-8 (a cyclic nucleotide specific inhibitor) had no effect. The effect of H-7 was dose dependent and present at varying concentrations of LPS. Down regulation of PKC activity by preincubation of KCs with phorbol myristate acetate (PMA, a direct activator of PKC) also resulted in significantly reduced TNF release after LPS stimulation. The inhibitor H-7 (10 microM) also significantly inhibited LPS signaled prostaglandin E2 release in Kupffer cells. Total and specific intracellular protein phosphorylation was determined by trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis after labeling stimulated Kupffer cells with 32Pi. Total protein phosphorylation was not significantly altered by LPS stimulation; however, autoradiograms from PMA- and LPS-stimulated KCs demonstrate enhanced phosphorylation of a 40-kDa protein (2.7 +/- 0.9-fold) and a 33-kDa protein (3.1 +/- 1.0-fold) which were inhibited by H-7. We conclude that activation of PKC and protein phosphorylation are required steps in the signal transduction pathway of LPS-stimulated TNF production in Kupffer cells.

Modulation of cytolytic T cell responses by heparan sulfate.

Accumulating evidence suggests that the functional properties of alloactivated T cells may depend upon the microenvironment in which the T cells reside. For instance, we showed previously that heparan sulfate, a biologically active polysaccharide present on cell surfaces and extracellular matrices, modulates the proliferative responses of splenocytes through enhancement of cytokine and prostaglandin production by macrophages. Here we report that under conditions of suboptimal stimulation, heparan sulfate causes discrete alterations in the functional responses of murine cytolytic T cells. When present in a 5-day mixed leukocyte culture (MLC), heparan sulfate mediates an increase, from 3- to 10-fold, in T cell-mediated cytotoxicity. This increase is dose dependent and most pronounced when heparan sulfate is present in the highest concentration during the first 24 hr of the culture period. On the other hand, when added during the last 48-72 hr of an MLC, heparan sulfate decreases cytotoxicity by 3- to 30-fold. Neutralizing antibodies against IL-1 alpha, but not antibodies against IL1 beta, IL-6, or TNF alpha/beta, abrogate the heparan sulfate-mediated increase in cytotoxicity, suggesting that the increase depended in part upon the production of IL-1 alpha. However, studies in which exogenous IL-1 was added to MLC showed that increased cytotoxicity was not due only to increased cytokine production. Augmentation of cytotoxicity was in part independent of T cell help, as depletion of CD4+ cells from the responder population before MLC, or addition of neutralizing anti-murine IL-2 antibodies plus human IL-2 to the MLC, did not abrogate the stimulatory effect of heparan sulfate. Heparan sulfate-treated CD8+ lymphoblasts isolated after 7 days in MLC demonstrated an increased cytotoxicity, elevated intracellular serine esterase, and perforin levels compared with lymphoblasts from control MLC. The decrease in cytotoxicity observed when heparan sulfate was present during the last several days of an MLC was likely mediated by PGE2, as elevated levels of PGE2 were detected in MLC supernatants of heparan sulfate-treated cultures, and because the decrease was not observed in the presence of indomethacin. Our results are consistent with the idea that the metabolism of heparan sulfate, an endogenous component of parenchymal tissues, may regulate the tempo and magnitude of alloreactive cytotoxic T cell responses.

Modulation of Kupffer cell membrane phospholipid function by n-3 polyunsaturated fatty acids

Dietary n-3 polyunsaturated fatty acids (PUFAs) have been reported to improve clinical outcome in a number of inflammatory diseases including burns and sepsis. One mechanism contributing to the anti-inflammatory effect is the incorporation of n-3 PUFAs into membrane phospholipids which decreases macrophage eicosanoid production. We hypothesize that an additional mechanism for their effects is an alteration of membrane signal transduction that decreases macrophage responsiveness to inflammatory stimuli. Kupffer cells, the fixed macrophages of the liver, were obtained from rats pair fed diets for 6 weeks with 15% of calories supplied as menhaden (high n-3), corn (control), or safflower (high n-6) oils. The effects of the dietary oils on Kupffer cell membrane signal transduction and eicosanoid production were assessed by measuring inositol phospholipid (PI) metabolism, intracellular calcium responses, and prostaglandin E2 (PGE2) production to the inflammatory signals endotoxin (LPS) and platelet activating factor (PAF). The menhaden oil diet resulted in significant incorporation of n-3 PUFAs into total cellular PUFAs compared to corn and safflower oil. (total n-3 PUFAs, 28.1% menhaden vs 2.1% corn vs 1.2% safflower, P less than 0.03). This incorporation altered signal transduction of PAF as both PI turnover (65% +/- 10% of corn oil) and calcium response (0.6-fold vs 5.0-fold for corn oil) were significantly reduced in the menhaden oil group. (P less than 0.05) The menhaden oil diet also reduced significantly PGE2 production in response to PAF and LPS (corn, 348 +/- 23 pg/ml; menhaden, 48 +/- 6 pg/ml, P less than 0.01). We conclude that, in addition to modulating eicosanoid production, n-3 PUFAs can also alter macrophage membrane signal transduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Sp proteins and Runx2 mediate regulation of matrix gla protein (MGP) expression by parathyroid hormone.

As part of its catabolic action in bone, parathyroid hormone (PTH) inhibits extracellular matrix mineralization. We previously showed that PTH dose‐dependently induces matrix gla protein (MGP) expression in osteoblasts and this induction is at least partially responsible for PTH‐mediated inhibition of mineralization. Recently, we identified PKA and ERK/MAPK as the key signaling pathways involved in PTH regulation of MGP expression. The goal of this study was to further characterize the mechanism by which PTH stimulates expression of MGP. Deletion analysis of the murine Mgp gene promoter identified a PTH‐responsive region between −173 bp and−49 bp. Using gel‐mobility shift assays we found that Sp1/Sp3, and Runx2 bind to distinct sites within this region. Mutation of either the Sp or the Runx2 site reduced MGP induction by PTH, while mutation of both sites completely abolished PTH responsiveness. Overexpression of Runx2 or Sp1 activated the Mgp reporter, while Sp3 was a dose‐dependent repressor of Sp1 and PTH‐induced MGP expression. Collectively, these data show that PTH regulates MGP gene transcription in osteoblasts through altered activities of Sp and Runx2 transcription factors. J. Cell. Biochem. 107: 284–292, 2009.

Regulation of matrix Gla protein by parathyroid hormone in MC3T3-E1 osteoblast-like cells involves protein kinase A and extracellular signal-regulated kinase pathways

Inhibition of osteoblast‐mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix γ‐carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH‐mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3‐E1 cells. Following transient transfection of these cells with a −748 bp murine MGP promoter‐luciferase construct (pMGP‐luc), PTH (10 −7 M) induced promoter activity in a time‐dependent manner with a maximal four‐ to six fold induction seen 6 h after PTH treatment. Both H‐89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co‐transfection of MC3T3‐E1 cells with pMGP‐luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose‐dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12‐myristate 13‐acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal‐regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA‐ and ERK‐dependent pathways. J. Cell. Biochem. 102: 496–505, 2007.