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Biomedical subjects

G Karsenty

Publications and source records attributed to G Karsenty.

At least 19 recordsLinked to original sources
PubMed2005

Osteopetrosis-like phenotype in latent TGF-beta binding protein 3 deficient mice.

LTBPs are extracellular matrix proteins resembling fibrillins. LTBP-1, 3, and 4 covalently bind latent TGF-beta and modulate tissue levels of this potent cytokine through regulation of its secretion, localization, and/or activation. To address LTBP function in vivo, we generated Ltbp-3 null mice. Ltbp-3-/- animals developed craniofacial abnormalities due to early ossification of the skull base synchondroses and displayed reduced body size. In addition, histological examination of Ltbp-3-/- skeletons revealed an increase in bone mass. The osteoblast numbers and mineral apposition rates were decreased in Ltbp-3-/- mice, whereas the osteoclast numbers were similar in null and wild type mice. Histological examination revealed persistence of cartilage remnants in Ltbp-3-/- trabecular bone. Taken together, these results indicate that the Ltbp-3-/- high bone mass phenotype was due to a defect in bone resorption. We hypothesize that lack of Ltbp-3 results in decreased levels of TGF-beta in bone and cartilage, which leads to compromised osteoclast function and decreased bone turnover.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Serum leptin level is a regulator of bone mass.

Leptin is a powerful inhibitor of bone formation in vivo. This antiosteogenic function involves leptin binding to its receptors on ventromedial hypothalamic neurons, the autonomous nervous system and beta-adrenergic receptors on osteoblasts. However, the mechanisms whereby leptin controls the function of ventromedial hypothalamic antiosteogenic neurons remain unclear. In this study, we compared the ability of leptin to regulate body weight and bone mass and show that leptin antiosteogenic and anorexigenic functions are affected by similar amounts of leptin. Using a knock-in of LacZ in the leptin locus, we failed to detect any leptin synthesis in the central nervous system. However, increasing serum leptin level, even dramatically, reduced bone mass. Conversely, reducing serum-free leptin level by overexpressing a soluble receptor for leptin increased bone mass. Congruent with these results, the high bone mass of lipodystrophic mice could be corrected by restoring serum leptin level, suggesting that leptin is an adipocyte product both necessary and sufficient to control bone mass. Consistent with the high bone mass phenotype of lipodystrophic mice, we observed an advanced bone age, an indirect reflection of premature bone formation, in lipodystrophic patients. Taken together, these results indicate that adipocyte-derived circulating leptin is a determinant of bone formation and suggests that leptin antiosteogenic function is conserved in vertebrates.

Animals↗
PubMed2004

Knock-in of nuclear localised beta-galactosidase reveals that the tyrosine phosphatase Ptprv is specifically expressed in cells of the bone collar.

Ptprv is a member of the transmembrane tyrosine phosphatase gene family reported to be expressed in osteoblasts and gonads. To better define the developmental and tissue specificity of Ptprv expression, we generated knock-in mice expressing a nuclear localised beta-galactosidase reporter under the control of resident Ptprv regulatory elements. Histochemical staining of Ptprv-nLacZ mice revealed that Ptprv expression is readily detectable in the foetal gonadal ridge of both sexes and in adult gonads where it is localised to Sertoli cells of the testis and celomic epithelial cells of the ovaries. During early limb development, Ptprv expression is prominent in the apical ectodermal ridge of the limb bud. At latter stages of development, Ptprv is predominantly expressed in the perichondrial and periosteal region of long bones, known as the bone collar. In contrast to previous indications from in vitro studies, there is little if any expression in mature osteoblasts in vivo. Analysis of Ptprv mRNA localisation by in situ hybridization in parallel with molecular markers of chondrocytes and osteoblasts confirmed the specific expression of Ptprv in immature bone collar cells. The specificity of Ptprv expression in these cells may be a useful tool to elucidate their role in the transition of skeletal elements from cartilage template to bone.

Animals↗
PubMed2004

[Bone mass regulation by leptin: a hypothalamic control of bone formation].

Bone mass is maintained constant between puberty and menopause by the balance between osteoblasts and osteoclasts activity. The existence of a hormonal control of osteoblast activity has been speculated for years by analogy to osteoclast biology. Through the search for such humoral signal(s) regulating bone formation, leptin has been identified as a powerful inhibitor of bone formation. Furthermore, by means of intracerebroventricular infusion of leptin, it has been shown that the effect of this adipocyte-derived hormone on bone is mediated via a brain relay, like all its other functions. Subsequent studies have led to the identification of hypothalamic neurons involved in leptin's antiosteogenic function. In addition, it has been shown that those neurons or neuronal pathways are distinct from neurons responsible for the regulation of energy metabolism. Finally, the peripheral mediator of leptin's antiosteogenic function has been identified as being the sympathetic nervous system. Catecholamine-deficient mice have a high bone mass and sympathomimetics administered to mice decreased bone formation and bone mass. Conversely, beta-blockers increased bone formation and bone mass and blunt the bone loss induced by ovariectomy.

Adipocytes↗
PubMed2004

Major complications of urinary diversion after pelvic exenteration for gynecologic malignancies: a 23-year mono-institutional experience in 124 patients.

OBJECTIVE: The objective of this study was to analyze the postoperative outcome of patients undergoing urinary diversion at the time of pelvic exenteration. METHODS: Between January 1980 and December 2002, 232 pelvic exenterations for gynecologic malignancies were performed in our hospital. One hundred and twenty-four included a urinary diversion. There were locally advanced or recurrent cancers including 101 cervical, 11 endometrial, 5 vagina, 2 ovarian malignancies and 5 pelvic sarcoma. RESULTS: Ninety patients (72.5%) had a history of previous irradiation. Exenterations were 69 anterior and 55 total. Urinary diversion included 14 bilateral ureterostomies, 62 trans-intestinal diversion and 48 continent diversion using distal ileum and right colon. Pelvic filling was performed in 56 patients (45%). Low colorectal anastomosis was performed in 42 of 48 supralevator pelvic exenteration (87.5%). Postoperative mortality rate was 8% (10/124). Overall 12-week postoperative morbidity rate was 52% (65/124) and appears to be significantly higher in irradiated patients and after total exenteration. In trans-intestinal noncontinent group, eight patients were reoperated for a complication directly related to urinary diversion procedure. No reoperation for such a complication was performed in the continent urinary diversion group. CONCLUSIONS: Ileocolic continent pouch seems to be the safer urinary diversion procedure after exenteration for gynecological malignancies especially in irradiated patients and after total exenteration.

Adult↗
PubMed2003

Cartilage formation and calcification in arteries of mice lacking matrix Gla protein.

Matrix Gla protein (MGP/Mgp) is a protein expressed predominantly by vascular smooth muscle cells (VSMCs) and by chondrocytes. Transgenic mice lacking Mgp die 1-3 months after birth due to calcification of elastic fibers and rupture of large elastic arteries such as the aorta. Here, we report on cartilage formation that commonly occurs in calcified arteries of Mgp-/- mice. Using histology, von Kossa staining, immunohistochemistry, and Western blotting, together with examination of cellular markers for VSMCs and extracellular matrix markers for cartilage, we provide evidence for cell transformation from VSMC to chondrocyte in the arterial media in the absence of Mgp. At 2 weeks of age in the aorta of Mgp-/- mice, VSMCs lose immunostaining for smooth muscle alpha-actin concomitant with the appearance of cartilage molecules as shown by immunohistochemical staining and Western blotting for aggrecan, link protein, and type II collagen. These data provide evidence that the absence of Mgp, and/or calcification of the ECM, in the arterial media can trigger chondrocyte differentiation and cartilage formation in blood vessels.

Actins↗
PubMed2002

Cbfa1 does not regulate RANKL gene activity in stromal/osteoblastic cells.

The rates of osteoblast and osteoclast formation are tightly balanced, possibly due to the requirement of mesenchymal osteoblast progenitors for osteoclastogenesis. Osteoblast differentiation requires the transcription factor Cbfa1, whereas osteoclastogenesis results from the interaction between receptor activator of NF kappa B ligand (RANKL), expressed on stromal/osteoblastic cells, and RANK, a surface receptor on hematopoietic precursors. A striking decrease in the number of osteoclasts in Cbfa1-deficient mice suggested that Cbfa1 might be involved in RANKL expression. To investigate this possibility and to elucidate the mechanisms regulating RANKL expression, we isolated the 5'-flanking region of the murine RANKL gene and found that it contains two potential binding sites for Cbfa1 (OSE2-like sites). Cbfa1 bound to either of these sites in gel shift assays and stimulated the activity of a chimeric promoter consisting of multimerized RANKL OSE2-like sites inserted upstream from a minimal thymidine kinase (tk) promoter in transient transfections. However, Cbfa1 cotransfection did not stimulate murine RANKL promoter-luciferase constructs. Further analysis revealed that removal of these sites from the RANKL promoter by either site-directed mutagenesis or 5'-deletion did not alter the basal activity of promoter-reporter constructs. Conditional expression of Cbfa1 in a stromal/osteoblastic cell line stimulated osteocalcin mRNA by fivefold, but had no significant effect on RANKL mRNA levels. Conversely, conditional expression of a dominant-negative form of Cbfa1 in the same cell line inhibited osteocalcin mRNA by threefold, but had no effect on RANKL mRNA. Although these results cannot rule out a novel function for Cbfa1 in RANKL expression, they demonstrate that Cbfa1 does not regulate RANKL gene activity in the same manner as known targets of this transcription factor, such as osteocalcin.

Amino Acid Sequence↗
PubMed2001

Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers.

Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, beta-glycerophosphate (betaGP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22alpha and smooth muscle alpha-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor alpha1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP(-/-)) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.

Animals↗
PubMed2001

YY1 is a positive regulator of transcription of the Col1a1 gene.

Both cell-specific and ubiquitous transcription factors in fibroblasts have been identified as critical for expression of the Col1a1 gene, which encodes the alpha1 chain of type I collagen. Here, we report that Yin Yang 1 (YY1) binds to the Col1a1 promoter immediately upstream of the TATA box, and we examine the functional implications of YY1 binding for regulation of Col1a1 gene expression in BALBc/3T3 fibroblasts. The Col1a1 promoter region spanning base pairs (bp) -56 to -9 bound purified recombinant YY1 and the corresponding binding activity in nuclear extracts was supershifted using a YY1-specific antibody. Mutation of the TATA box to TgTA enhanced YY1 complex formation. Mutation analysis revealed two YY1 core binding sites at -40/-37 bp (YY1A) and, on the reverse strand, at -32/-29 bp (YY1B) immediately adjacent to the TATA box. In transfections using Col1a1-luciferase constructs, mutation of YY1A decreased activity completely (wild-type p350 (p350wt), -222/+113 bp) or partially (p130wt, -84 bp/+13 bp), whereas mutation of YY1B blocked the expression of both promoter constructs. Cotransfection with pCMV-YY1 increased p350wt and p130wt activities by as much as 10-fold, whereas antisense YY1 decreased constitutive expression and blocked the increased activity due to pCMV-YY1 overexpression. The mTgTA constructs were devoid of activity, arguing for a requirement for cognate binding of the TATA box-binding protein (TBP). Electrophoretic mobility shift assays performed under conditions permitting TBP binding showed that recombinant TBP/TFIID and YY1 could bind to the -56/-9 bp fragment and that YY1B was the preferred site for YY1 binding. Our results indicate that YY1 binds to the Col1a1 proximal promoter and functions as a positive regulator of constitutive activity in fibroblasts. Although YY1 is not sufficient for transcriptional initiation, it is a required component of the transcription machinery in this promoter.

3T3 Cells↗
PubMed2001

Prostate cancer cells induce osteoblast differentiation through a Cbfa1-dependent pathway.

Metastases from prostatic adenocarcinoma (prostate cancer) are characterized by their predilection for bone and typical osteoblastic features. An in vitro model of bone metastases from prostate cancer was developed using a bicompartment coculture system of mouse osteoblasts and human prostate cancer cells. In this model, the bone-derived prostate cancer cell lines MDA PCa 2a and MDA PCa 2b induced a specific and reproducible increase in osteoblast proliferation. Moreover, these cells were able to induce osteoblast differentiation, as assessed by increased alkaline phosphatase activity, Osteocalcin expression, and calcified matrix formation. This osteoblastic reaction was confirmed in vivo by intrafemoral injection of MDA PCa 2b cells into severe combined immunodeficiency disease mice. In contrast, the highly undifferentiated, bone-derived human prostate cancer cell line PC3 did not produce an osteoblastic reaction in vitro and induced osteolytic lesions in vivo. The osteoblast differentiation induced by MDA PCa 2b cells was associated with up-regulation of the osteoblast-specific transcriptor factor Cbfa1. Moreover, treatment of osteoblasts with conditioned medium obtained from MDA PCa 2b cells resulted in up-regulation of Cbfa1 and Osteocalcin expression. In support of the differentiation studies, a microarray analysis showed that primary mouse osteoblasts grown in the presence of MDA PCa 2b cells showed a shift in the pattern of gene expression with an increase in mRNA-encoding Procollagen type I and Osteopontin and a decrease in mRNA-encoding proteins associated with myoblast differentiation, namely myoglobin and myosin light-chain 2. Taken together, these findings suggest that the bone-derived prostate cancer cells MDA PCa 2a and MDA PCa 2b promote differentiation of osteoblast precursors to an osteoblastic phenotype through a Cbfa1-dependent pathway. These results also established that soluble factors produced by prostate cancer cells can induce expression of osteoblast-specific genes. This in vitro model provides a valuable system to isolate molecules secreted by prostate cancer cells that favor osteoblast differentiation. Moreover, it allows to screen for therapeutic agents blocking the osteoblast response to prostate cancer.

Animals↗
PubMed2001

TGF-beta-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation.

Transforming growth factor-beta (TGF-beta), a secreted factor present at high levels in bone, inhibits osteoblast differentiation in culture; yet, the mechanism of this inhibition remains unclear. We studied the effects of TGF-beta and its effectors, the Smads, on the expression and function of the osteoblast transcription factor CBFA1. TGF-beta inhibited the expression of the cbfa1 and osteocalcin genes, whose expression is controlled by CBFA1 in osteoblast-like cell lines. This inhibition was mediated by Smad3, which interacts physically with CBFA1 and represses its transcriptional activity at the CBFA1-binding OSE2 promoter sequence. The repression of CBFA1 function by Smad3 contrasts with previous observations that Smads function as transcription activators. This repression occurred in mesenchymal but not epithelial cells, and depended on the promoter sequence. Smad3-mediated repression of CBFA1 provides a central regulatory mechanism for the inhibition of osteoblast differentiation by TGF-beta, since it inhibits both cbfa1 transcription and transcriptional activation of osteoblast differentiation genes by CBFA1. Altering Smad3 signaling influenced osteoblast differentiation in the presence or absence of TGF-beta, implicating Smad3/TGF-beta-mediated repression in autocrine regulation of osteoblast differentiation.

Animals↗
PubMed2001

Continuous expression of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice.

Chondrocyte hypertrophy is a mandatory step during endochondral ossification. Cbfa1-deficient mice lack hypertrophic chondrocytes in some skeletal elements, indicating that Cbfa1 may control hypertrophic chondrocyte differentiation. To address this question we generated transgenic mice expressing Cbfa1 in nonhypertrophic chondrocytes (alpha1(II) Cbfa1). This continuous expression of Cbfa1 in nonhypertrophic chondrocytes induced chondrocyte hypertrophy and endochondral ossification in locations where it normally never occurs. To determine if this was caused by transdifferentiation of chondrocytes into osteoblasts or by a specific hypertrophic chondrocyte differentiation ability of Cbfa1, we used the alpha1(II) Cbfa1 transgene to restore Cbfa1 expression in mesenchymal condensations of the Cbfa1-deficient mice. The transgene restored chondrocyte hypertrophy and vascular invasion in the bones of the mutant mice but did not induce osteoblast differentiation. This rescue occurred cell-autonomously, as skeletal elements not expressing the transgene were not affected. Despite the absence of osteoblasts in the rescued animals there were multinucleated, TRAP-positive cells resorbing the hypertrophic cartilage matrix. These results identify Cbfa1 as a hypertrophic chondrocyte differentiation factor and provide a genetic argument for a common regulation of osteoblast and chondrocyte differentiation mediated by Cbfa1.

Animals↗
PubMed2001

Central control of bone formation.

Vertebrates constantly remodel bone to maintain a constant bone mass. Bone remodeling comprises two phases: bone resorption by the osteoclasts followed by bone formation by the osteoblasts. Although the prevailing view about the control of bone remodeling is that it is an autocrine/paracrine phenomenon, the bone resorption arm of bone remodeling is under a tight endocrine control. To date little is known about the regulation of bone formation. We took the observations that gonadal failure favors bone loss and obesity protects from it as an indication that bone mass, body weight, and reproduction could be regulated by the same hormone(s). Leptin is one of these hormones. Leptin inhibits bone formation by the osteoblasts. This function is dominant, and leptin deficiency results in a high bone mass phenotype despite the hypogonadism characterizing these animals. Genetic biochemical and physiological studies demonstrate that leptin inhibits bone formation following its binding to its receptor in the hypothalamus. These results are the first evience that bone remodeling is a hypothalamic process; they imply necessarily that osteoporosis, the most frequent bone remodeling disease, is partly at least a hypothalamic disease. This finding also has therapeutic implications.

Animals↗