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Biomedical subjects

S L Teitelbaum

Publications and source records attributed to S L Teitelbaum.

At least 19 recordsLinked to original sources
PubMed2003

ATM variants 7271T>G and IVS10-6T>G among women with unilateral and bilateral breast cancer.

Recent reports suggest that two ATM gene mutations, 7271T>G and IVS10-6T>G, are associated with a high risk of breast cancer among multiple-case families. To assess the importance of these two mutations in another 'high-risk' group, young women (under age 51) with multiple primaries, we screened a large population-based series of young women with bilateral breast cancer and compared the frequency of these mutations among similar women diagnosed with unilateral breast cancer. The 1149 women included were enrolled in an ongoing population-based case-control study of the genetic factors that contribute to bilateral breast cancer; they were not selected on the basis of family history of cancer. Screening for 7271T>G and IVS10-6T>G ATM gene mutations was conducted using DHPLC followed by direct sequencing. The 7271T>G mutation was detected in one out of 638 (0.2%) women with unilateral breast cancer and in none of the bilateral cases, and the IVS10-6T>G mutation in one out of 511 (0.2%) bilateral and in eight out of 638 (1.3%) unilateral breast cancer cases. Carriers of either mutation were not limited to women with a family history. Given the likelihood that young women with bilateral breast cancer have a genetic predisposition, the observed mutation distribution is contrary to that expected if these two mutations were to play an important role in breast carcinogenesis among individuals at high risk.

Adult↗
PubMed2002

Abnormal osteoclast morphology and bone remodeling in a murine model of a lysosomal storage disease.

Mucopolysaccharidosis type VII (MPS VII) is a heritable lysosomal storage disease caused by a deficiency in beta-glucuronidase (GUSB) activity, leading to progressive accumulation of undegraded glycosaminoglycans in many tissues. Clinical features include growth and mental retardation, hearing and visual defects, shortened lifespan, and skeletal deformities. A murine model of MPS VII has been described that shares many of the manifestations of the human disease, including the skeletal dysplasia. In this study we describe abnormalities in the cellular morphology and function of osteoclasts and a localized defect in bone formation rate in the MPS VII mouse. Ultrastructural analysis revealed that MPS VII osteoclasts fail to form ruffled border membranes and many appeared to be detached from the bone surface. Following bone marrow transplantation, osteoclasts derived from wild-type donors showed normal morphology and were closely associated with the bone surface in MPS VII recipients. In vitro bone resorption assays demonstrated that MPS VII osteoclasts formed significantly smaller and fewer pits than those formed by osteoclasts derived from normal mice of the same strain. Although osteoclast morphology and function appeared to be abnormal in the MPS VII mouse, interleukin-1 (IL-1)-induced osteoclastogenesis in vivo was not affected. In addition to the osteoclast defects, MPS VII mice demonstrated a slower rate of bone matrix deposition in the epiphysis by in vivo calcein labeling experiments. These data suggest that abnormal morphology and function of MPS VII osteoclasts, combined with deficient matrix deposition, may contribute to the skeletal defects observed in this lysosomal storage disease.

Animals↗
PubMed2001

Rho family GTPases regulate VEGF-stimulated endothelial cell motility.

Migration of endothelial cells induced by vascular endothelial growth factor (VEGF) is a critical step in angiogenesis. Stimulation of motility by growth factors such as VEGF requires interaction with the signal transduction pathways activated by the extracellular matrix (ECM). Here we demonstrate that the Rac GTPase is the critical intersection activated by type 1 collagen ECM and VEGF during stimulation of endothelial cell motility. To analyze the role of the Rho family GTPases in VEGF-stimulated endothelial cell chemotaxis and ECM-stimulated haptotaxis, we transduced the respective fusion proteins in human foreskin dermal endothelial cells using a Tat peptide from the human immunodeficiency virus Tat protein. VEGF signaling required Rac activation during chemotaxis, and Rac and Cdc42 were activated during haptotaxis on type I collagen. Similar to VEGF, Rac activation induced an increase in endothelial cell stress fiber and focal adhesion. Surprisingly, Rho activation was not present in collagen-induced haptotaxis or stimulation of chemotaxis by VEGF, although Rho induced stress fibers and focal adhesions similar to Rac activation. The result of constitutive Rho activation was an inhibition of haptotaxis. Thus, Rac is required and sufficient for the activation of endothelial cell haptotaxis and VEGF-stimulated chemotaxis.

Actins↗
PubMed2001

TAT fusion proteins containing tyrosine 42-deleted IkappaBalpha arrest osteoclastogenesis.

In most circumstances, NF-kappaB, which is essential for osteoclastogenesis, is activated following serine 32/36 phosphorylation of its cytosolic inhibitory protein, IkappaBalpha. In contrast to other cell types, IkappaBalpha, in bone marrow macrophages (BMMs), which are osteoclast precursors, is tyrosine-phosphorylated by c-Src kinase. To address the role of IkappaBalpha phosphorylation in osteoclastogenesis, we generated TAT fusion proteins containing wild-type IkappaBalpha (TAT-WT-IkappaB), IkappaBalpha lacking its NH(2)-terminal 45 amino acids (TAT-IkappaB(46-317)), and IkappaBalpha in which tyrosine residue 42, the c-Src target, is mutated into phenylalanine (TAT-IkappaB(Y42F)). TAT-IkappaB efficiently enters BMMs, and the NF-kappaB-inhibitory protein, once intracellular, is functional. While TAT-WT-IkappaB only slightly inhibits osteoclastogenesis, osteoclast recruitment is diminished >80% by TAT-IkappaB(46-317), an event mirrored by dentin resorption. The fact that TAT alone does not impact osteoclastogenesis, which also resumes following withdrawal of TAT-IkappaB(46-317), establishes that the mutant's anti-osteoclastogenic properties do not reflect toxicity. Affirming a functional role for IkappaB(Tyr(42)) in osteoclastogenesis, TAT-IkappaB(Y42F) is as efficient as TAT-IkappaB(46-317) in blocking osteoclast differentiation. Thus, dominant-negative IkappaBalpha constructs block osteoclastogenesis, and Tyr(42) is essential to the process, increasing the possibility that nonphosphorylatable forms of IkappaBalpha may be a means of preventing pathological bone loss.

Animals↗
PubMed2001

Cloning and characterization of the murine beta(3) integrin gene promoter: identification of an interleukin-4 responsive element and regulation by STAT-6.

Expression of the alpha(v)beta(3) integrin by murine bone marrow macrophages is regulated by cytokines such as IL-4 and GM-CSF through transcriptional activation of the beta(3) subunit gene. To characterize the molecular mechanisms by which such regulation occurs, we isolated the murine beta(3) integrin promoter. To this end, we first cloned a full length beta(3) cDNA and used the 5'UTR and leader peptide coding sequence to identify genomic clones containing the beta(3) promoter region. The transcriptional start site, identified by primer extension and S1 nuclease assay, is 34 nt upstream of the translation initiation codon. A 1.1 kb fragment of the promoter region drives IL-4 responsive transcription in transiently transfected murine bone marrow macrophages. Deletion analysis of the beta(3) promoter indicates the IL-4 responsive element lies between -465 to -678 nt relative to the transcriptional start site. This promoter fragment contains two overlapping STAT consensus recognition sites and nuclear extracts from BMMs contain an IL-4-inducible DNA binding factor, identified by super shift analysis, as STAT-6. Furthermore, an oligonucleotide which includes the two STAT recognition sites residing in the IL-4 responsive region of the beta(3) promoter, competes for STAT-6 binding. Confirming IL-4 induction of the integrin subunit is specifically mediated by STAT-6, beta(3) mRNA is not enhanced in BMMs derived from STAT-6 deleted mice, which however, retain their capacity to respond to GM-CSF.

5' Untranslated Regions↗
PubMed2001

A Glanzmann's mutation in beta 3 integrin specifically impairs osteoclast function.

Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the alpha v beta 3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the beta 3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of beta 3(-/-) osteoclast function following expression of a full-length beta 3 integrin. In contrast, truncated beta 3, lacking a cytoplasmic domain (h beta 3c), is completely ineffective in restoring function to beta 3(-/-) osteoclasts. To identify the components of the beta 3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate beta integrin signaling. Of the six, only the S(752)P substitution, which also characterizes a form of the human bleeding disorder Glanzmann's thrombasthenia, fails to rescue beta 3(-/-) osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y(747)F/Y(759)F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the beta 3 integrin regulates platelets and osteoclasts.

Amino Acid Sequence↗
PubMed2001

Crystal structure of the TRANCE/RANKL cytokine reveals determinants of receptor-ligand specificity.

RANK, the receptor activator of NF-kappaB, and its ligand RANKL (initially termed TRANCE, also termed ODF and OPGL), are a TNF superfamily receptor-ligand pair that govern the development and function of osteoclasts, lymphoid tissue, and mammary epithelium. While TNF family cytokines share a common structural scaffold, individual receptor-ligand pairs associate with high specificity. Given the low level of amino acid conservation among members of the TNF superfamily, the means by which these molecules achieve specificity cannot be completely understood without knowledge of their three-dimensional structures. To determine the elements of RANKL that mediate RANK activation, we have crystallized the ectodomain of murine RANKL and solved its structure to a resolution of 2.6 A. RANKL self-associates as a homotrimer with four unique surface loops that distinguish it from other TNF family cytokines. Mutagenesis of selected residues in these loops significantly modulates RANK activation, as evidenced by in vitro osteoclastogenesis, thereby establishing their necessity in mediating the biological activities of RANKL. Such structural determinants of RANKL-RANK specificity may be of relevance in the pharmacologic design of compounds to ameliorate osteopenic disorders of bone.

Animals↗
PubMed2001

Receptor activator of nuclear factor-kappa b ligand activates nuclear factor-kappa b in osteoclast precursors.

Receptor activator of nuclear factor-kappa B ligand [RANK ligand (RANK-L)] stimulates mature osteoclasts to resorb bone, a process associated with NF-kappa B activation. RANK-L also prompts macrophages to develop the osteoclast phenotype. Although NF-kappa B is essential for osteoclast differentiation, it is not known whether RANK-L activates this transcription complex in osteoclast precursors. We report that RANK-L rapidly induces NF-kappa B activation in both authentic osteoclast precursors, namely bone marrow macrophages, and RAW 264.7 cells, a murine macrophage line also capable of RANK-L-mediated osteoclastogenesis. Supershift studies reveal the RANK-L-induced DNA binding moiety contains p50/p65, the most common NF-kappa B complex. Subcellular translocation of p50 and p65 subunits is confirmed by Western blots and immunofluorescence analysis. RANK-L activates NF-kappa B in both bone marrow macrophages and RAW 264.7 cells by serine phosphorylation of I kappa B alpha within 5 min, resulting in rapid I kappa B alpha degradation and resynthesis. Attesting to function, RANK-L treatment of RAW 264.7 cells transiently transfected with a plasmid containing NF-kappa B consensus elements linked to luciferase greatly enhances reporter activity. Our data suggest that activation of the NF-kappa B pathway is an integral component of RANK-L-induced osteoclast differentiation.

Animals↗
PubMed2000

Transforming growth factor-beta up-regulates the beta 5 integrin subunit expression via Sp1 and Smad signaling.

Integrin-mediated cell-matrix interactions play important roles in regulating cell function. Since transforming growth factor-beta (TGF-beta) modulates many osteoblast activities, we hypothesized that the growth factor acts in part by modulating integrin expression. TGF-beta increased cell adhesion to vitronectin and up-regulated the surface level of alpha(v)beta(5) via increasing beta(5) protein synthesis by a transcriptional mechanism. Promoter activity analysis demonstrated that a TGF-beta-responsive element resides between nucleotides -63 and -44. Electrophoretic mobility shift assay and immunoprecipitation/Western studies indicated that the nuclear complex formed using the -66/-42 oligonucleotide contained both Sp1/Sp3 and Smad proteins. Since nuclear Sp1/Sp3 levels were not altered, whereas Smad levels were increased by TGF-beta, we investigated the roles of Smad proteins in the up-regulation of beta(5) gene activation. Co-transfection of cells with beta(5) promoter reporter construct and expression vectors for Smad3, Smad4, and Sp1 increased the stimulatory effect of TGF-beta. Furthermore, expression of dominant negative Smad3 or Smad4 in cells decreased or abolished the stimulation of beta(5) promoter activity by TGF-beta. Smad4 mutant also inhibited the up-regulation of surface beta(5) level by TGF-beta. Thus, TGF-beta increases expression of the integrin beta(5) gene by mechanisms involving Sp1/Sp3 and Smad transcription factors.

Animals↗
PubMed2000

Tumor necrosis factor receptors types 1 and 2 differentially regulate osteoclastogenesis.

The potent osteoclastogenic agent, tumor necrosis factor-alpha (TNF), exerts its biological effects via two receptors, namely TNF receptors 1 (p55r) and 2 (p75r), each present on osteoclast precursors. Thus, we asked if p55r and p75r differentially impact the osteoclastogenic process. Marrow derived from mice expressing only p55r generates substantially more osteoclasts, in the basal state, than does wild type, while marrow expressing only p75r, produces substantially fewer. Reflecting its preferential activation of p55r, exogenous TNF stimulates osteoclast formation by p55r(+/+)p75r(-/-), but not p55r(-/-)p75r(+/+), marrow. Consistent with the fact that NF-kappaB is essential for osteoclastogenesis, this transcription complex is activated, relative to wild type, in p55r(+/+)p75r(-/-) osteoclast precursors and suppressed in those expressing only p75r. Because p55r enhances, and p75r suppresses, osteoclastogenesis, we asked if their principal ligands, namely soluble and membrane-residing TNF, respectively, differentially impact basal osteoclast recruitment. We find, in contrast to the significant level of osteoclast formation in wild type marrow, osteoclastogenesis by that derived from mice expressing membrane, but not soluble, TNF, is negligible. Thus, optimal therapeutic inhibition of bone resorption may entail selective TNF receptor modulation and/or arrested cleavage of membrane TNF to its soluble form.

Animals↗
PubMed2000

Bone resorption by osteoclasts.

Osteoporosis, a disease endemic in Western society, typically reflects an imbalance in skeletal turnover so that bone resorption exceeds bone formation. Bone resorption is the unique function of the osteoclast, and anti-osteoporosis therapy to date has targeted this cell. The osteoclast is a specialized macrophage polykaryon whose differentiation is principally regulated by macrophage colony-stimulating factor, RANK ligand, and osteoprotegerin. Reflecting integrin-mediated signals, the osteoclast develops a specialized cytoskeleton that permits it to establish an isolated microenvironment between itself and bone, wherein matrix degradation occurs by a process involving proton transport. Osteopetrotic mutants have provided a wealth of information about the genes that regulate the differentiation of osteoclasts and their capacity to resorb bone.

Animals↗
PubMed2000

Immunohistochemical analysis of polycyclic aromatic hydrocarbon-DNA adducts in breast tumor tissue.

Environmental carcinogens may play a role in the etiology of breast cancer, but the extent of their contribution is not yet defined. The aims of this study were to determine whether polycyclic aromatic hydrocarbon (PAH)-DNA adducts could be detected in stored paraffin blocks of breast tumor tissue (n=147) with an immunoperoxidase technique and whether they correlated with smoking history and/or mutant p53 protein expression. There was no significant difference in mean relative nuclear staining intensity in non-smokers (444+/-90, n=75), ever smokers (435+/-91, n=72), and current smokers (456+/-98, n=35). In either current or ever smokers, PAH-DNA adducts were non-significantly elevated in those with greater compared with lower exposure in relation to age at started smoking, years of smoking, cigarettes per day, and pack years. DNA damage levels were not elevated in tissues with compared with those without mutant p53 protein expression. These data demonstrate that immunohistochemical methods can be used to monitor DNA damage levels in archived breast tissues.

Adult↗
PubMed2000

Sp1/Sp3 and PU.1 differentially regulate beta(5) integrin gene expression in macrophages and osteoblasts.

Murine osteoclast precursors and osteoblasts express the integrin alpha(v)beta(5), the appearance of which on the cell surface is controlled by the beta(5), and not the alpha(v), subunit. Here, we show that a 173-base pair proximal region of the beta(5) promoter mediates beta(5) basal transcription in macrophage (osteoclast precursor)-like and osteoblast-like cells. DNase I footprinting reveal four regions (FP1-FP4) within the 173-base pair region, protected by macrophage nuclear extracts. In contrast, osteoblast nuclear extracts protect only FP1, FP2, and FP3. FP1, FP2, and FP3 bind Sp1 and Sp3 from both macrophage and osteoblast nuclear extracts. FP4 does not bind osteoblast proteins but binds PU.1 from macrophages. Transfection studies show that FP1 and FP2 Sp1/Sp3 sites act as enhancers in both MC3T3-E1 (osteoblast-like) and J774 (macrophage-like) cell lines, whereas the FP3 Sp1/Sp3 site serves as a silencer. Mutation of the FP2 Sp1/Sp3 site totally abolishes promoter activity in J774 cells, with only partial reduction in MC3T3-E1 cells. Finally, we demonstrate that PU.1 acts as a beta(5) silencer in J774 cells but plays no role in MC3T3-E1 cells. Thus, three Sp1/Sp3 sites regulate beta(5) gene expression in macrophages and osteoblast-like cells, with each element exhibiting cell-type and/or activation-suppression specificity.

Animals↗
PubMed2000

Accelerated bone mineral loss in HIV-infected patients receiving potent antiretroviral therapy.

BACKGROUND: The use of highly active antiretroviral therapy (HAART) has been associated with multiple metabolic complications whose pathogenesis is poorly understood at the present time. METHODS: We performed a cross-sectional analysis of whole-body, lumbar spine (L1-L4) and proximal femur bone mineral density in 112 male subjects (HIV-infected patients on HAART that included a protease inhibitor, HIV-infected patients not receiving a protease inhibitor and healthy seronegative adults) using dual energy x-ray absorptiometry. RESULTS: Men receiving protease inhibitors had a higher incidence of osteopenia and osteoporosis according to World Health Organization definitions: relative risk = 2.19 (95% confidence interval 1.13-4.23) (P = 0.02). Subjects receiving protease inhibitors had greater central: appendicular adipose tissue ratios than the other two groups (P < 0.0001). There was no relationship between the central: appendicular fat ratio and the lumbar spine or proximal femur bone mineral density t- or z- scores, suggesting that osteoporosis and body fat redistribution are independent side effects of HAART. CONCLUSIONS: Osteopenia and osteoporosis are unique metabolic complications associated with protease inhibitor-containing potent antiretroviral regimens, that appear to be independent of adipose tissue maldistribution.

Absorptiometry, Photon↗
PubMed2000

Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells.

Long-term administration of pharmacological doses of glucocorticoids inhibits bone formation and results in osteoporosis. Since integrin-mediated cell-matrix interactions are essential for osteoblast function, we hypothesized that the detrimental effect of glucocorticoids on bone derived, at least in part, from decreased integrin-matrix interactions. Because alphavbeta3 and alphavbeta5 integrins can interact with several bone matrix proteins, we analyzed the effects of dexamethasone (Dex) on the expression of these integrins in normal human osteoblastic cells. We found adhesion of these cells to osteopontin and vitronectin to be dependent on alphavbeta3 and alphavbeta5, respectively; this ligand specificity was not altered by Dex. The effects of Dex on the adhesion of human osteoblastic cells to osteopontin and vitronectin were biphasic with an increase after 2 days, followed by a decrease after 8 days of treatment. Consistently, surface alphavbeta3 and alphavbeta5 integrins, which were increased after 2 days of Dex treatment, were decreased after 8 days. Similarly, total cellular alphav, beta3, and beta5 proteins, which were increased by Dex early in the culture, were diminished after 8 days. Metabolic labeling studies indicated that Dex exhibited biphasic regulation on the biosynthesis of alphavbeta5, with stimulation observed during the second day of treatment, followed by inhibition during the 8th day of exposure. By contrast, the biosynthesis of alphavbeta3 was inhibited by Dex on day 1 and remained inhibited on day 8. Analysis of the mRNA indicated that alphav and beta5 levels were increased by Dex during early exposure (1-3 days), followed by inhibition after prolonged exposure (>/=7 days). By contrast, Dex decreased beta3 mRNA level at all the time points analyzed. Consistently, Dex decreased beta3 promoter activity after 1 day and persisted over 8-day period. By contrast, Dex stimulated beta5 promoter activity after 1 or 2 days but had no effect after 8 days. To further evaluate mechanism(s) leading to the decreased integrin expression after prolonged Dex treatment, mRNA stability was analyzed. Dex was found to accelerate the degradation of alphav, beta3 and beta5 mRNA after an 8-day treatment. Thus, the regulation of alphavbeta3 was dependent on transcription and posttranscriptional events whereas the expression of alphavbeta5 was dependent mainly on posttranscriptional events after prolonged Dex treatment. In conclusion, Dex exhibited time-dependent regulation on the expression of alphavbeta3 and alphavbeta5 integrins in normal human osteoblastic cells. Short-term exposure to Dex increased the levels of alphavbeta3 and alphavbeta5 on the surface and cell adhesion to osteopontin and vitronectin whereas long-term exposure to Dex decreased the expression of both integrins and inhibited the cell adhesion to matrix proteins.

Antigens, CD↗
PubMed2000

Mice deficient in Abl are osteoporotic and have defects in osteoblast maturation.

The c-Abl protein is a non-receptor tyrosine kinase involved in many aspects of mammalian development. c-Abl kinase is widely expressed, but high levels are found in hyaline cartilage in the adult, bone tissue in newborn mice, and osteoblasts and associated neovasculature at sites of endochondrial ossification in the fetus. Mice homozygous for mutations in the gene encoding c-Abl (AIM) display increased perinatal mortality, reduced fertility, foreshortened crania and defects in the maturation of B cells in bone marrow. Here we demonstrate that Abl-/- mice are also osteoporotic. The long bones of mutant mice contain thinner cortical bone and reduced trabecular bone volume. The osteoporotic phenotype is not due to accelerated bone turnover--both the number and activity of osteoclasts are similar to those of control littermates--but rather to dysfunctional osteoblasts. In addition, the rate of mineral apposition in the mutant animals is reduced. Osteoblasts from both stromal and calvarial explants showed delayed maturation in vitro as measured by expression of alkaline phosphatase (ALP), induction of mRNA encoding osteocalcin and mineral deposition.

Alkaline Phosphatase↗