Biomedical subjects

Laura R McCabe

Publications and source records attributed to Laura R McCabe.

15 recordsLinked to original sources

PubMed2006

Bone loss and increased bone adiposity in spontaneous and pharmacologically induced diabetic mice.

Insulin-dependent diabetes mellitus (IDDM) is associated with increased risk of osteopenia/osteoporosis in humans. The mechanisms accounting for diabetic bone loss remain unclear. Pharmacologic inducers of IDDM, such as streptozotocin, mimic key aspects of diabetes in rodents, allow analysis at the onset of diabetes, and induce diabetes in genetically modified mice. However, side effects of streptozotocin, unrelated to diabetes, can complicate data interpretation. The nonobese diabetic (NOD) mouse model develops diabetes spontaneously without external influences, negating side effects of inducing agents. Unfortunately, in this model the onset of diabetes is unpredictable, occurs in a minority of male mice, and can only be studied in a single mouse strain. To validate the relevance of the more flexible streptozotocin-induced diabetes model for studying diabetes-associated bone loss, we compared its phenotype to the spontaneously diabetic NOD model. Both models exhibited hyperglycemia and loss of body, fat pad, and muscle weight. Furthermore, these genetically different and distinct models of diabetes induction demonstrated similar bone phenotypes marked by significant trabecular bone loss and increased bone marrow adiposity. Correspondingly, both diabetic models exhibited decreased osteocalcin mRNA and increased adipocyte fatty acid-binding protein 2 mRNA levels in isolated tibias and calvaria. Taken together, multiple streptozotocin injection-induced diabetes is a valid model for understanding the acute and chronic pathophysiologic responses to diabetes and their mechanisms in bone.

Adipose Tissue↗

PubMed2006

Chronic hyperglycemia modulates osteoblast gene expression through osmotic and non-osmotic pathways.

Insulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with osteoporosis and increased fracture rates. The mechanisms underlying IDDM associated bone loss are not known. Previously we demonstrated that osteoblasts exhibit a response to acute (1 and 24 h) hyperglycemia and hyperosmolality. Here we examined the influence of chronic hyperglycemia (30 mM) and its associated hyperosmolality on osteoblast phenotype. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions (mannitol treatment). Acute hyperglycemia for a 48-h period was also capable of inducing alkaline phosphatase and suppressing osteocalcin, MMP-13, VEGF, and GAPDH expression in differentiated osteoblasts. This suggests that acute responses in differentiated cells are maintained chronically. In addition, hyperglycemic and hyperosmotic conditions increased PPARgamma2 expression, although this increase reached significance only in 21 days chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARgamma2 expression is increased in type I diabetic mouse model bones, these findings suggest that diabetes-associated hyperglycemia may modulate osteoblast gene expression, function and bone formation and thereby contribute to type I diabetic bone loss.

Animals↗

PubMed2006

Normal bone density obtained in the absence of insulin receptor expression in bone.

Type I diabetes is characterized by little or no insulin production and hyperglycemic conditions. It is also associated with significant bone loss and increased bone marrow adiposity. To examine the role of reduced insulin signaling in type I diabetic bone loss without inducing hyperglycemia, we used genetically reconstituted insulin receptor knockout mice (IRKO-L1) that are euglycemic as a result of human insulin receptor transgene expression in the pancreas, liver, and brain. RT-PCR analyses demonstrated undetectable levels of insulin receptor expression in IRKO-L1 bone, yet IRKO-L1 bones exhibit similar (and trend toward greater) bone density compared with wild-type animals as determined by microcomputed tomography. More detailed bone analyses indicated that cortical bone area was increased in tibias of IRKO-L1 mice. Osteoblast markers (osteocalcin and runx2 mRNA levels) and resorption markers (serum pyridinoline levels) were similar in wild-type and IRKO-L1 bones. When marrow adiposity was examined, we noticed a decrease in adipocyte number and fatty-acid-binding protein 2 expression in IRKO-L1 mice compared with wild-type mice. Bone marrow stromal cell cultures obtained from wild-type and IRKO-L1 mice demonstrated similar adipogenic and osteogenic potentials, indicating that systemic factors likely contribute to differences in marrow adiposity in vivo. Interestingly, IGF-I receptor mRNA levels were elevated in IRKO-L1 bones, suggesting (in combination with hyperinsulinemic conditions) that increased IGF-I receptor signaling may represent a compensatory response and contribute to the changes in cortical bone. Taken together, these results suggest that reduced insulin receptor signaling in bone is not a major factor contributing to bone loss in type I diabetes.

Adipocytes, White↗

PubMed2006

Inhibition of PPARgamma prevents type I diabetic bone marrow adiposity but not bone loss.

Diabetes type I is associated with bone loss and increased bone adiposity. Osteoblasts and adipocytes are both derived from mesenchymal stem cells located in the bone marrow, therefore we hypothesized that if we could block adipocyte differentiation we might prevent bone loss in diabetic mice. Control and insulin-deficient diabetic BALB/c mice were chronically treated with a peroxisomal proliferator-activated receptor gamma (PPARgamma) antagonist, bisphenol-A-diglycidyl ether (BADGE), to block adipocyte differentiation. Effects on bone density, adiposity, and gene expression were measured. BADGE treatment did not prevent diabetes-associated hyperglycemia or weight loss, but did prevent diabetes-induced hyperlipidemia and effectively blocked diabetes type I-induced bone adiposity. Despite this, BADGE treatment did not prevent diabetes type I suppression of osteoblast markers (runx2 and osteocalcin) and bone loss (as determined by micro-computed tomography). BADGE did not suppress osteoblast gene expression or bone mineral density in control mice, however, chronic (but not acute) BADGE treatment did suppress osteocalcin expression in osteoblasts in vitro. Taken together, our findings suggest that BADGE treatment is an effective approach to reduce serum triglyceride and free fatty acid levels as well as bone adiposity associated with type I diabetes. The inability of BADGE treatment to prevent bone loss in diabetic mice suggests that marrow adiposity is not linked to bone density status in type I diabetes, but we cannot exclude the possibility of additional BADGE effects on osteoblasts or other bone cells, which could contribute to preventing the rescue of the bone phenotype.

Adipocytes↗

PubMed2005

Increased bone adiposity and peroxisomal proliferator-activated receptor-gamma2 expression in type I diabetic mice.

Decreased bone mass, osteoporosis, and increased fracture rates are common skeletal complications in patients with insulin-dependent diabetes mellitus (IDDM; type I diabetes). IDDM develops from little or no insulin production and is marked by elevated blood glucose levels and weight loss. In this study we use a streptozotocin-induced diabetic mouse model to examine the effect of type I diabetes on bone. Histology and microcomputed tomography demonstrate that adult diabetic mice, exhibiting increased plasma glucose and osmolality, have decreased trabecular bone mineral content compared with controls. Bone resorption could not completely account for this effect, because resorption markers (tartrate-resistant acid phosphatase 5b, urinary deoxypyridinoline excretion, and tartrate-resistant acid phosphatase 5 mRNA) are unchanged or reduced at 2 and/or 4 wk after diabetes induction. However, osteocalcin mRNA (a marker of late-stage osteoblast differentiation) and dynamic parameters of bone formation were decreased in diabetic tibias, whereas osteoblast number and runx2 and alkaline phosphatase mRNA levels did not differ. These findings suggest that the final stages of osteoblast maturation and function are suppressed. We also propose a second mechanism contributing to diabetic bone loss: increased marrow adiposity. This is supported by increased expression of adipocyte markers [peroxisome proliferator-activated receptor gamma2, resistin, and adipocyte fatty acid binding protein (alphaP2)] and the appearance of lipid-dense adipocytes in diabetic tibias. In contrast to bone marrow, adipose stores at other sites are depleted in diabetic mice, as indicated by decreased body, liver, and peripheral adipose tissue weights. These findings suggest that IDDM contributes to bone loss through changes in marrow composition resulting in decreased mature osteoblasts and increased adipose accumulation.

Adipose Tissue↗

PubMed2005

Adsorption of serum fetuin to hydroxylapatite does not contribute to osteoblast phenotype modifications.

Osteoblasts exhibit enhanced differentiation and altered gene profiles when cultured on hydroxyapatite (HA) compared to plastic surfaces. To begin determining mechanisms for this response, we used proteomics to identify proteins predominantly found in osteoblasts on HA but not plastic surfaces. Two-dimensional gel electrophoresis and Western analyses indicate that fetuin is abundant in extracts from HA, but not plastic surfaces. Incubation of HA and plastic surfaces with cell culture medium (containing 10% serum) under cell-free conditions shows that fetuin is predominantly derived from the culture medium serum and readily adsorbs to the HA surface. However, we did detect low levels of fetuin B mRNA in osteoblasts. Serum albumin, actin-beta, apolipoprotein-AI, and vimentin also adsorbed to HA. To determine the role of fetuin in the HA-induced osteoblast phenotype changes, osteoblasts were seeded onto fetuin-coated or uncoated HA under serum-free conditions. Osteoblast morphology was similar on both HA surfaces, suggesting that HA alone (without adsorbed serum proteins) is sufficient for cell attachment and spreading. Similarly, genes previously reported to be modulated by HA (glvr-1, DMP-1, osteoglycin, and proliferin 3) were modulated even in the absence of fetuin or other serum proteins. These data show that HA surface can be enriched selectively with fetuin from serum; however, neither fetuin or other serum proteins are required to mediate HA-induced osteoblast attachment, spreading, or changes in expression of genes examined. This finding suggests that factors intrinsic to HA are required for the response.

Adsorption↗

PubMed2005

Biaxial flexure testing of calcium phosphate bioceramics for use in tissue engineering.

This study analyzes data from 206 CaP specimens (68 HA, 70 BCP, and 68 beta-TCP) fractured via biaxial flexure testing. Specimens were divided into four groups: (a) Group I, dry; (b) Group II, wet (day 0, immersion time approximately 5-10 s); (c) Group III, after immersion in media for 21 days (day 21); and (d) Group IV, after culturing osteoblasts (OBs) on the surface for 21 days (day 21 with cells). X-ray diffraction verified the presence of minor second phases in HA and beta-TCP while BCP was a biphasic mixture of HA and beta-TCP with minor phases present. The statistical significance (p < 0.05) of differences in the measured biaxial flexure fracture strength, S, between groups was assessed via one-way ANOVA with Tukey's test. Also, a two-parameter Weibull analysis assessed the mechanical reliability of each group. Osteoblasts increase the biaxial flexure fracture strength in a statistically significant way compared to both the HA discs in Groups II and III. Scanning electron microscope examination revealed grain boundary grooving on the sintered surfaces and with thermal expansion anisotropy, likely leads to the observed rapid strength decline upon exposure to media found in Groups II, III and IV.

Animals↗

PubMed2004

Inhibition of cross-bridge formation has no effect on contraction-associated phosphorylation of p38 MAPK in mouse skeletal muscle.

Mitogen-activated protein kinases (MAPKs), in particular p38 MAPK, are phosphorylated in response to contractile activity, yet the mechanism for this is not understood. We tested the hypothesis that the force of contraction is responsible for p38 MAPK phosphorylation in skeletal muscle. Extensor digitorum longus (EDL) muscles isolated from adult male Swiss Webster mice were stimulated at fixed length at 10 Hz for 15 min and then subjected to Western blot analysis for the phosphorylation of p38 MAPK and ERK1/2. Contralateral muscles were fixed at resting length and were not stimulated. Stimulated muscles showed a 2.5-fold increase in phosphorylated p38 MAPK relative to nonstimulated contralateral controls, and there was no change in the phosphorylation of ERK1/2. When contractile activity was inhibited with N-benzyl-p-toluene sulfonamide (BTS), a specific inhibitor of actomyosin ATPase, force production decreased in both a time- and concentration-dependent manner. Preincubation with 25, 75, and 150 microM BTS caused 78+/-4%, 97+/-0.2%, and 99+/-0.2% inhibition in contractile force, respectively, and was stable after 30 min of treatment. Fluorescence measurements demonstrated that Ca2+ cycling was minimally affected by BTS treatment. Surprisingly, BTS did not suppress the level of p38 MAPK phosphorylation in stimulated muscles. These data do not support the view that force generation per se activates p38 MAPK and suggest that other events associated with contraction must be responsible.

Animals↗

PubMed2004

Osteoblasts respond to hydroxyapatite surfaces with immediate changes in gene expression.

Bone mineral contains hydroxyapatite (HA). This is the surface that mature osteoblasts and osteocytes interact with. Synthetic HA is widely used in orthopedic surgeries as an implant or implant coating. The bone-like HA surfaces increase implant union and bone formation; however, the mechanisms accounting for this effect on osteoblasts are not known. In this study, we compared gene expression profiles of osteoblasts responding to HA or plastic surfaces for 24 h. Expression profiles were also compared between HA discs processed with gravity-sieved compared with combined gravity and air-jet-sieved HA powders. The latter, composed of smaller HA particles, exhibits an increase in grain boundary surface area. Discs made with either HA powder similarly up-regulated osteoblast expression of 10 genes (including proliferin 3, Glvr-1, DMP-1, and tenascin C) and down-regulated 15 genes (such as osteoglycin) by more than 2-fold compared with plastic surfaces. The overall changes are indicative of an immediate (24-h) response to the HA surface and a trend toward osteoblast differentiation. In addition, subsets of modulated genes exist that are unique to each HA subtype. Taken together, we identified HA responsive genes evident within 24 h of surface contact, indicating a critical role for extracellular mineral surfaces in the regulation of osteoblast gene expression and phenotype.

3T3 Cells↗

PubMed2004

CCAAT/enhancer-binding protein-beta has a role in osteoblast proliferation and differentiation.

CCAAT/enhancer binding protein beta (C/EBPbeta) is known to play an important role in the expression of several genes necessary for bone development and homeostasis including osteocalcin, IGF-1, and IL-6. In this study, we show that C/EBPbeta protein levels and, consequently, DNA-binding activity are temporally regulated, dramatically decreasing upon differentiation of MC3T3-E1 mouse osteoblasts. Corresponding with these results, the constitutive expression of C/EBPbeta LAP in MC3T3-E1 osteoblasts increased proliferation and suppressed osteogenic differentiation. Thus, C/EBPbeta LAP not only appears to participate in the regulation of genes associated with mature bone physiology, but is also a critical regulator of osteoblast growth and differentiation.

3T3 Cells↗

PubMed2004

Hypoxia suppresses runx2 independent of modeled microgravity.

Bone loss is a consequence of skeletal unloading as seen in bed rest and space flight. Unloading decreases oxygenation and osteoblast differentiation/function in bone. Previously we demonstrated that simulation of unloading in vitro, by culturing differentiating mouse osteoblasts in a horizontal rotating wall vessel (RWV), results in suppressed expression of runx2, a master transcriptional regulator of osteoblast differentiation. However, the RWV is able to reproduce in a controlled fashion at least two aspects of disuse that are directly linked, model microgravity and hypoxia. Hypoxia in the RWV is indicated by reduced medium oxygen tension and increased expression of GAPDH and VEGF. To uncouple the role of model microgravity from hypoxia in suppressed runx2 expression, we cultured osteoblasts under modeled microgravity (oxygenated, horizontal RWV rotation), hypoxia (vertical RWV rotation), or both conditions (horizontal RWV rotation). The expression, DNA binding activity and promoter activity of runx2, was suppressed under hypoxic but not normoxic modeled microgravity RWV conditions. Consistent with a role for hypoxia in suppression of runx2, direct exposure to hypoxia alone is sufficient to suppress runx2 expression in osteoblasts grown in standard tissue culture plates. Taken together, our findings indicate that hypoxia associated with skeletal unloading could be major suppressor of runx2 expression leading to suppressed osteoblast differentiation and bone formation.

3T3 Cells↗

PubMed2003

Simulated microgravity suppresses osteoblast phenotype, Runx2 levels and AP-1 transactivation.

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood but involve the suppression of osteoblast function. To elucidate the influence of mechanical unloading on osteoblasts, a rotating wall vessel (RWV) was employed as a ground based model of simulated microgravity. Mouse MC3T3-E1 osteoblasts were grown on microcarrier beads for 14 days and then placed in the RWV for 24 h. Consistent with decreased bone formation during actual spaceflight conditions, alkaline phosphatase and osteocalcin expression were decreased by 80 and 50%, respectively. In addition, runx2 expression and AP-1 transactivation, key regulators of osteoblast differentiation and bone formation, were reduced by more than 60%. This finding suggests that simulated microgravity could promote dedifferentiation and/or transdifferentiation to alternative cell types; however, markers of adipocyte, chondrocyte, and myocyte lineages were not induced by RWV exposure. Taken together, our results indicate that simulated microgravity may suppress osteoblast differentiation through decreased runx2 and AP-1 activities.

3T3 Cells↗

PubMed2003

Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant-negative connexin 43.

Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB-F344 rat liver epithelial or mouse osteoblastic MC3T3-E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY-457) or LY with a MW of 649 Da (LY-649). Transfection of a dominant negative-connexin 43 (Cx43) gene decreased GJIC (>50%) when LY-649 was used, however, normal GJIC was observed in both cell lines when LY-457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3-E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3-E1. WB-F344 cells normally exhibit a biphasic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network-like structures by WB-F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild-type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals.

Animals↗

PubMed2003

Adrenomedullin increases AP-1 expression in rat mesangial cells via activation of protein kinase-A and p38 MAPK.

Adrenomedullin (AM), a potent vasodilatory peptide, has anti-proliferative and pro-apoptotic effects in rat mesangial cells (RMCs). We have previously demonstrated that AM modulates activities of the members of MAPK family in RMCs. Because activation of MAPKs has been reported to induce AP-1 expression in other cell systems, the major aim of the present study was to examine the effects and mechanisms of AM on AP-1 member mRNA expression, in RMCs. AM produced a concentration- and time-dependent increase in c-fos and jun-B mRNA expression without observable effects on fos-B and c-jun. Associated with this change, AM increased the AP-1 DNA binding activity. RMCs transiently transfected with a luciferase fusion gene driven by an AP-1 cis-element demonstrate a concentration- and time-dependent increase in luciferase activity suggesting that AM increases AP-1-mediated gene transcription in these cultured cells. Both an AM-induced increase in AP-1 mRNA expression and AP-1 luciferase activities were inhibited by H89 (protein kinase-A inhibitor) and SB203580 (p38 MAPK inhibitor). These results indicate a likely role for the members of AP-1 family in AM-mediated biological responses in rat mesangial cells and a role for cAMP activation and subsequent activation of p38 MAPK in AM-induced AP-1 activity.

Adrenomedullin↗

PubMed2002

P38 and activating transcription factor-2 involvement in osteoblast osmotic response to elevated extracellular glucose.

Poorly controlled or untreated type I diabetes mellitus is characterized by hyperglycemia and is associated with decreased bone mass and osteoporosis. We have demonstrated that osteoblasts are sensitive to hyperglycemia-associated osmotic stress and respond to elevated extracellular glucose or mannitol by increasing c-jun and collagen I expression. To determine whether MAPKs are involved in this response, MC3T3-E1 osteoblasts were treated with 16.5 mm glucose, mannitol, or contrast dye for 1 h. Immunoblotting of phosphorylated p38 demonstrated activation of p38 MAPK by hyperosmotic stress in vitro and in vivo. Activation peaked at 20 min, remained detectable after 24 h, and was protein kinase C-independent. Activating transcription factor-2 (ATF-2) activation followed the same pattern as phospho-p38. Transactivation of cAMP response element (CRE)- and c-jun promoter (containing a CRE-like element)-reporter constructs increased following hyperosmotic treatment. SB 203580 (a p38 MAPK inhibitor) blocked ATF-2 phosphorylation, CRE transactivation, and c-jun promoter activation. Hyperosmotic activation of collagen I promoter activity was also inhibited by SB 203580, consistent with the involvement of c-jun in collagen I up-regulation. Therefore, we propose that hyperglycemia-induced increases in p38 MAPK activity and ATF-2 phosphorylation contribute to CRE activation and modulation of c-jun and collagen I expression in osteoblasts.

3T3 Cells↗