Biomedical subjects

J M Gimble

Publications and source records attributed to J M Gimble.

At least 37 records · Page 2Linked to original sources

PubMed1998

1,25-Dihydroxy vitamin D3 inhibits adipocyte differentiation and gene expression in murine bone marrow stromal cell clones and primary cultures.

Bone marrow stromal stem cells differentiate into adipocytes and osteoblasts. These two lineages are thought to be reciprocally related, in part due to the observation that the osteoblast-inducing factor, 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], inhibited adipogenesis of rat femoral-derived stromal cell cultures. However, the literature is divided concerning the adipogenic effects of this steroid hormone. This work examined the effect of 1,25(OH)2D3 (10(-12)-10(-8) M) on murine femoral-derived bone marrow stromal cell differentiation in response to adipogenic agonists employing two different classes of nuclear hormone receptors: the glucocorticoid receptor (hydrocortisone) or peroxisome proliferator-activated receptors (thiazolidinediones). Experiments used the multipotent murine bone marrow stromal cell line, BMS2, and its subclones, as well as primary-derived murine bone marrow stromal cell cultures. In all systems examined, 1,25(OH)2D3 blocked adipogenesis induced by hydrocortisone, methylisobutylxanthine, and indomethacin based on flow cytometric analysis of lipid accumulation. This correlated with reduced messenger RNA levels of the late adipocyte gene markers, aP2 and adipsin. In the BMS2 subclone no. 24, the 1,25(OH)2D3 actions were concentration dependent. Whereas 1,25(OH)2D3 partially inhibited thiazolidinedione-induced adipogenesis in the parental BMS2 cell line, it had minimal effect on the thiazolidinedione-induced differentiation of the BMS2 subclone and primary cultures. These findings indicate that 1,25(OH)2D3, at nanomolar concentrations, completely inhibits murine bone marrow stromal cell differentiation in response to glucocorticoid-based adipogenic agonists but is a less effective adipogenic antagonist following induction with thiazolidinediones. This work supports the conclusion that 1,25(OH)2D3 inhibits murine femoral-derived bone marrow stromal cell adipogenesis.

1-Methyl-3-isobutylxanthine↗

PubMed1998

The derivation and characterization of stromal cell lines from the bone marrow of p53-/- mice: new insights into osteoblast and adipocyte differentiation.

We have derived a series of clonal cell lines from the bone marrow of p53-/- mice that represent different stages of osteoblast and adipocyte differentiation. All cell lines show indefinite growth potential (>300 population doublings) and have generation times of 12-20 h. These cell lines have been grouped into three categories. The least mature clones are heterogeneous and appear to contain a subpopulation of stem cells, which can spontaneously generate foci that contain either adipocytes or mineralizing osteoblasts. The second category of clones are homogeneous and clearly correspond to mature osteoblasts because they express high levels of the anticipated osteoblastic markers in a stable fashion and cannot differentiate into adipocytes even in the presence of inducers. The clones in the third category are the most unique. Initially they appeared to correspond to mature osteoblasts because they express alkaline phosphatase in a homogeneous manner, secrete type I collagen, show a significant cyclic adenosine monophosphate response to parathyroid hormone, secrete osteocalcin, and mineralize extensively after only 4-7 days. However, in contrast to the mature osteoblasts, these clones can be induced to undergo massive adipocyte differentiation, and this differentiation is accompanied by the complete loss of expression of all osteoblastic markers except alkaline phosphatase. These observations indicate that some cells that have acquired all of the characteristics of mature osteoblasts can be diverted to the adipocyte pathway. Further characterization of these clones may be particularly relevant to osteoporotic conditions where increased adipocyte formation appears to occur at the expense of osteoblast formation.

Adipocytes↗

PubMed1998

Nuclear hormone receptors and adipogenesis.

Adipocytes provide a model cell system for exploring the complexities of nuclear hormone receptor transcriptional regulation. Adipocytes produce lipid and cholesterol metabolites which can serve as activating ligands for many of the classic and "orphan" nuclear hormone receptors. At the same time, nuclear hormone receptors directly control adipocyte commitment. The recently described nuclear hormone receptor coactivators and corepressors provide an additional level of complexity to this system. This review emphasizes available in vitro and in vivo models and discusses them in the context of current controversies and future experimental directions.

Adipose Tissue↗

PubMed1997

CAAT/enhancer binding proteins directly modulate transcription from the peroxisome proliferator-activated receptor gamma 2 promoter.

The CCAAT/enhancer binding proteins (C/EBPs) and the peroxisome proliferator-activated receptors (PPARs) together regulate adipogenesis. The current work uses co-transfection studies to examine the C/ EBP dependence of PPAR gamma 2 transcription. Both C/ EBP alpha and C/EBP delta expression vectors activated transcription from a PPAR gamma 2 promoter/luciferase expression vector by 5-6 fold in UMR106 cells. The simultaneous transfection of the C/EBP homologous protein (CHOP) (also known as growth arrest DNA damage protein 153 or gadd153) inhibited this C/EBP-dependent activation in a concentration dependent manner. The CHOP protein is known to heterodimerize with other C/EBP proteins to form transcriptionally inactive complexes. Mutation of the two C/EBP DNA recognition elements at -340 bp and -327 bp within the PPAR gamma 2 promoter reduced the inductive effects of both C/EBP alpha and C/EBP delta. These findings demonstrate that proteins within the C/EBP family directly modulate transcription from the PPAR gamma 2 promoter.

Animals↗

PubMed1997

Decreased expression of murine PPARgamma in adipose tissue during endotoxemia.

Infection-induced hyperlipidemia develops due to a combination of factors, one of which is decreased clearance of lipids from the bloodstream due to depressed synthesis of lipoprotein lipase (LPL). Recently, the peroxisome proliferator activated receptors (PPARs) have been shown to be important in the regulation of LPL, particularly PPARgamma. PPARgamma and its heterodimerization partner, RXR alpha have been shown to be transcriptional activators of LPL in co-transfection analysis. Therefore, we hypothesized that the decrease in LPL expression during endotoxemia may be a result of depressed PPARgamma expression. In these studies, we examined the effect of endotoxin or its proximal mediator, tumor necrosis factor (TNF), on the expression of PPARgamma in white (WAT) and brown adipose tissue (BAT) in CD-1 mice. We report that treatment with endotoxin, but not TNF, transiently decreased PPARgamma mRNA levels 4 hr after treatment. However, endotoxin or TNF treatment decreased PPARgamma protein levels after 18 hr, which was at a time when LPL mRNA levels were also depressed. These data suggest that decreased PPARgamma expression following endotoxin or TNF treatment may contribute to the hyperlipidemia due to decreased expression of LPL, which would impair triglyceride clearance.

Adipose Tissue↗

PubMed1996

Antibody ligation of CD9 modifies production of myeloid cells in long-term cultures.

The KMC8.8 monoclonal antibody was made by immunizing rats with the BMS2 stromal cell clone, and was selected for further study because its ability to inhibit production of myeloid cells in Dexter cultures but not that of lymphoid cells in Whitlock-Witte cultures. The influence on myeloid progenitors might have been indirect, since the antibody did not prevent responsiveness to colony-stimulating factors in semisolid agar cultures. Furthermore, there was no inhibition, and some augmentation, of cell production when the antibody was added to established Dexter cultures. A cDNA clone that encoded the KMC8.8-recognized molecule was isolated by expression cloning and found to be identical in sequence to a previously published murine CD9 homologue. The antibody and cDNA clone were used to establish that CD9 is expressed by stromal cells, megakaryocytes, platelets, myeloid cells, and subpopulations of mature lymphocytes in mice. Treatment with the KMC8.8/CD9 antibody slightly augmented adhesion between myeloid cells and stromal cells, consistent with previous reports that this member of the tetraspan family of proteins can transmit proadhesive signals to human platelets and lymphoid cells. CD9 might participate in cell-cell interactions critical for correct orientation and movement of maturing myeloid cells in bone marrow.

Animals↗

PubMed1996

Analysis of the native murine bone morphogenetic protein serine threonine kinase type I receptor (ALK-3).

The bone morphogenetic proteins, members of the transforming growth factor-beta cytokine family, induce the osteoblast phenotype and promote osteogenesis in the bone marrow stroma. Simultaneously, these cytokines inhibit other mesodermal differentiation pathways, such as adipogenesis and myogenesis. The receptors for the bone morphogenetic proteins belong to a family of transmembrane serine/ threonine kinase TGF beta type I and type II receptor proteins. In man, these include the activin receptor like kinase-3 (ALK-3), a type I receptor protein. We have used a polyclonal antibody to examine the expression of the native murine ALK-3 protein in murine tissues and bone morphogenetic protein-responsive cell lines. On Western blot analyses, we found that the native 85 kDa native ALK-3 protein was expressed in a number of murine tissues; protein and mRNA levels did not necessarily correlate. Two bone morphogenetic protein-responsive cell lines, BMS2 bone marrow stromal cells and C2C12 myoblasts, expressed the ALK-3 protein constitutively. Cell differentiation was accompanied by modest changes in ALK-3 protein levels. Immunoprecipitation of the ALK-3 protein cross linked to [125I] BMP-4 revealed two major receptor complexes of approximately 90 kDa and 170 kDa in size. Biotin surface-labeling experiments revealed that the 85 kDa ALK-3 protein was constitutively associated with a novel 140 kDa surface glycoprotein. Deglycosylation reduced the protein's size to 116 kDa, comparable in size to that of the recently described BMP type II receptor. These findings support the current model that BMP interacts with a pre-existing complex consisting of a type I and type II receptor protein.

Animals↗

PubMed1996

The function of adipocytes in the bone marrow stroma: an update.

The adipocyte is the most abundant stromal cell phenotype in adult human bone marrow. Four hypotheses may explain their function. First, adipocytes may serve a passive role, simply occupying excess space in the bone marrow cavity. Second, they may play an active role in systemic lipid metabolism. Third, adipocytes may provide a localized energy reservoir in the bone marrow. Or fourth, marrow adipocytes may contribute directly to the promotion of hematopoiesis and influence osteogenesis. This article reviews recent findings concerning bone marrow adipocyte morphology and physiology, the transcriptional and cytokine mechanisms regulating their differentiation, and the interrelationships existing between bone marrow adipocytes, hematopoiesis, and osteogenesis. Overall, these data lend support to a "plastic" model of bone marrow stromal cell differentiation; adipocytes may share common functions with stromal stem cells, osteoblasts, and hematopoietic supportive cells.

Adipocytes↗

PubMed1996

Prolactin receptor expression during adipocyte differentiation of bone marrow stroma.

To determine the role of the hormone prolactin and its receptor on the differentiation, growth, and metabolic activity of cells of bone marrow origin, prolactin receptor expression was assessed in bone marrow stromal cells. Using reverse transcription - polymerase chain reaction, BMS2 cells, a bone marrow stromal cell line, were shown to express prolactin receptors following adipocyte differentiation, using three different adipocyte-differentiation protocols. Primary bone marrow stromal cells also show a dose-dependent increase in prolactin receptor expression following treatment with adipogenic agonists. That prolactin receptor expression is inducible upon adipocyte differentiation was confirmed using a preadipocyte cell line 3T3 - L1. Further, prolactin receptor parallels lipoprotein lipase gene expression in 3T3-L1 cells. These results suggest that prolactin and its receptor may play a role in differentiation and/or metabolism of pre-adipocytes and adipocytes.

Adipocytes↗

PubMed1996

Peroxisome proliferator-activated receptor-gamma activation by thiazolidinediones induces adipogenesis in bone marrow stromal cells.

The thiazolidinediones improve insulin sensitivity in animal models and have promise as potent oral antidiabetic agents. Their clinical use has been limited because of the resulting anemia and cardiac hypertrophy. Some compounds of this class have been reported to induce bone marrow fat accumulation in animals, and this effect could account for the observed anemia. We examined the biological mechanism contributing to this phenomenon. The thiazolidinediones BRL49653 and pioglitazone induced adipocyte differentiation in the BMS2 bone marrow stromal cell line in a dose- and time-dependent manner. These actions were further enhanced by the presence of glucocorticoids and other adipogenic agonists. The thiazolidinediones increased the mRNA levels of adipocyte-specific genes, including that of their receptor, the peroxisome proliferator-activated receptor-gamma (PPAR gamma). In contrast, mRNA levels of genes encoding other PPAR family members (PPAR alpha, PPAR delta, or NUC-1) were unchanged or decreased. Thiazolidinedione treatment of primary bone marrow stromal cells elicited a comparable dose-dependent response. Using a polyclonal antibody, PPAR gamma was detected in protein lysates from adipose-rich bone marrow. Thus, thiazolidinedione directly regulates bone marrow stromal cell differentiation; induced PPAR gamma expression may play a key regulatory role in this process.

Adipocytes↗

PubMed1995

Cloning and characterization of the murine activin receptor like kinase-1 (ALK-1) homolog.

Multiple serine/threonine kinases were detected in a bone marrow stromal cell line. One of these, the murine activin receptor like kinase-1 (ALK-1) homolog was cloned and sequenced. The expressed recombinant protein (62 kDa) was consistent in size with that predicted by the cDNA (58.6 kDa). On Western blots, a goat polyclonal antibody detected the native ALK-1 protein in bone marrow stromal cells, lung, brain, kidney and spleen. Two protein species of 60 kDa and 72-76 kDa were detected. Glycosylation events or alternative splicing may account for the larger protein specie.

Activin Receptors↗

PubMed1995

COOH-terminal disruption of lipoprotein lipase in mice is lethal in homozygotes, but heterozygotes have elevated triglycerides and impaired enzyme activity.

The role of the enzyme lipoprotein lipase (LPL) in atherosclerosis is uncertain. To generate an animal model of LPL deficiency, we targeted the LPL gene in embryonic stem cells with a vector designed to disrupt the COOH terminus of the protein and used these cells to generate LPL-deficient mice. Germ line transmission of the disrupted LPL allele was achieved with two chimeric males, and offspring from each of these animals were phenotypically identical. Pups homozygous (-/-) for LPL deficiency died within 48 h of birth with extreme elevations of serum triglycerides (13,327 mg/dl) associated with essentially absent LPL enzyme activity in heart and carcass. Newborn heterozygous (+/-) LPL-deficient pups had lower LPL enzyme activity and higher triglycerides (370 versus 121 mg/dl) than wild type (+/+) littermates. Adult heterozygotes had higher triglycerides than wild type mice with ad libitum feeding (236 mg/dl for +/- versus 88 mg/dl for +/+) and after fasting for 4 h (98 mg/dl for +/- versus 51 for +/+) or 12 h (109 mg/dl for +/- versus 56 mg/dl for +/+). Triglycerides were present as very low density lipoprotein particles and chylomicrons, but high density lipoprotein cholesterol levels were not decreased in +/- animals. Plasma heparin-releasable LPL activity was 43% lower in +/- versus +/+ adult animals. LPL activity, mRNA, and protein were lower in the tissues of +/- versus +/+ mice. Homozygous LPL deficiency caused by disruption of the COOH terminus of the enzyme is lethal in mice. Heterozygous LPL deficiency caused by this mutation is associated with mild to moderate hypertriglyceridemia without affecting static HDL cholesterol levels. Heterozygous LPL-deficient mice could be useful for determining if hypertriglyceridemia, independently or in combination with other discrete defects, influences atherosclerosis.

Animals↗

PubMed1995

Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells.

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-beta gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.

Adipose Tissue↗

PubMed1995

Lipopolysaccharide regulation of lipoprotein lipase expression in murine macrophages.

The enzyme lipoprotein lipase is expressed in a number of cell types and plays a central role in lipid metabolism. Multiple factors regulate its expression in a tissue-specific manner. In murine macrophages, lipopolysaccharide inhibits lipoprotein lipase enzyme activity. The current work examines this process in the established J774 macrophage line and primary peritoneal macrophages from endotoxin-sensitive (C3HeB/Fej) and endotoxin-resistant (C3H/Hej) murine strains. Lipopolysaccharide inhibition of macrophage lipoprotein lipase occurred at the enzyme and mRNA levels in a time- and concentration-dependent manner. Cells from endotoxin-resistant animals maintained their expression of lipoprotein lipase following treatment with lipopolysaccharide. Results of gel retention assays showed that lipopolysaccharide treatment of the J774 macrophages altered the level of nuclear proteins recognizing and binding the lipoprotein lipase promoter DNA. Nuclear extracts from resting J774 cells contained proteins which bound specifically to the octamer motif and to the CAAT box within the lipoprotein lipase promoter. Exposure of the J774 cells to lipopolysaccharide for 16 h increased the level of protein-octamer DNA complexes. Similar responses were obtained in endotoxin-sensitive, but not endotoxin-resistant, primary macrophages following in vitro treatment with lipopolysaccharide. This finding suggests that transcriptional events may contribute to the lipopolysaccharide regulation of macrophage lipoprotein lipase expression.

Animals↗

PubMed1995

In vitro and in vivo analysis of murine lipoprotein lipase gene promoter: tissue-specific expression.

Lipoprotein lipase, an enzyme of central importance to lipid metabolism, is most abundant in adipose tissues, cardiac and skeletal muscle, and portions of the brain. The current work examined the murine lipoprotein lipase promoter using transient transfection, gel-retention analyses, and transgenic mice. Maximum expression of the luciferase reporter gene in transfected cells was observed with -101 bp of the promoter. Nuclear extracts from tissues expressing lipoprotein lipase contained DNA binding proteins that recognize the CCAAT box (-64 bp) and an octamer motif (-46 bp); this combination of factors was absent in nonexpressing tissues. Transgenic mice from three of five founders prepared with -1,824-bp promoter constructs expressed the luciferase reporter gene at highest levels in brown adipose tissue and brain. These findings suggest that the -1,824-bp promoter region contains sequence elements responsible for the tissue-specific transcription of lipoprotein lipase in vivo.

Animals↗

PubMed1994

Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein.

The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone-induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor alpha protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.

Adipocytes↗

PubMed1994

Recognition of murine integrin beta 1 by a rat anti-stromal cell monoclonal antibody.

Previous studies with the rat monoclonal antibody KMI6 had localized its antigen in vivo to a discrete subpopulation of marrow stromal cells. The KMI6 antigen has now been identified as the murine homolog of integrin beta 1 by amino acid sequence analysis and by cross-reactivity with antiserum to the avian integrin beta 1. The relative tissue abundance of murine integrin beta 1 was determined by Western blot. Although immunoperoxidase staining of fixed murine hematopoietic tissues demonstrated an abundance of intracellular beta 1, few primary-derived cells of lymphohematopoietic origin were surface positive as assessed by immunofluorescence and flow cytometry. Fetal erythroblasts provided the only exception. In contrast, the antigen was readily detected on the surface of several cultured cell lines in association with a variety of alpha chains. The biochemical properties of the surface labeled murine integrin beta 1 were similar to those of its human counterpart, exhibiting an altered electrophoretic migration under reduced conditions or following N-glycanase treatment. The antibody recognition of the protein was insensitive to glycosylation state, presence of divalent cations, detergents, or transfer to a nitrocellulose membrane. However, on Western blot, the epitope was lost on reduction of the protein, suggesting that it is conformation dependent. These data indicate that although KMI6 epitope is widely distributed, its surface expression in vivo may be restricted within lymphohemopoietic tissues.

Amino Acid Sequence↗

PubMed1994

Dimethyl sulfoxide modulates NF-kappa B and cytokine activation in lipopolysaccharide-treated murine macrophages.

Antioxidants are protective against septic shock in animal models. Recently, free radical scavengers have been found to inhibit the activation of the NF-kappa B protein in a number of cell lines. This transcriptional regulatory protein binds to the promoters of the proinflammatory cytokines tumor necrosis factor, interleukin-6, and the macrophage inflammatory proteins. The current work examined lipopolysaccharide-induced NF-kappa B activation in the J774 macrophage-like cell line and primary peritoneal macrophages from lipopolysaccharide-responsive (C3HeB/Fej) and -nonresponsive (C3H/HeJ) murine strains. The DNA-binding activity of the NF-kappa B protein directly correlated with mRNA expression for the genes encoding the proinflammatory cytokines and the free radical scavenging enzyme, superoxide dismutase. Both the p50 and p65 NF-kappa B subunits were detected on gel supershift assays. Minimal NF-kappa B activity was observed following exposure of C3H/HeJ macrophages to lipopolysaccharide. The antioxidant dimethyl sulfoxide decreased the level of NF-kappa B activation in the J774 cells. This correlated with decreased expression of cytokine mRNAs and tumor necrosis factor bioactivity. These results suggest that modulation of NF-kappa B activation may provide a mechanism through which antioxidants protect against endotoxemia in murine models.

Animals↗