Biomedical subjects

J Shen

Publications and source records attributed to J Shen.

At least 199 records · Page 11Linked to original sources

PubMed2000

[Expression and deletion analysis of EcoR II endonuclease and methylase gene].

OBJECTIVE: To clone complete EcoR II restriction endonuclease gene (ecoR II R) and methyl-transferase (ecoR II M) gene into one vector and to analyzing the expression of the whole system. METHODS: Unidirective deletion subclones constructed with Exo III, ecoR II R/M genes were preliminarily located in the cloned fragments according to the enzyme activities of each subclone, exact deletion sites were determined by sequencing, and transcriptional start sites were mapped by S1 mapping. RESULTS: The DNA fragment which was cloned into pBluescript SK+ contained the complete ecoR II R gene and ecoR II M gene, there are two transcriptional start sites in ecoR II R gene, 132 bp to 458 bp from 3' and of ecoR II R gene are indispensable to enzyme activities and deletion of 202 bp from 3' end of ecoR II M gene made it lose the capability to resist specific cut of EcoR II R enzyme, deletion of coding region and flanking sequence of one gene did not affect the expression of the other gene, the recombinant only containing ecoR II R gene appeared to be lethal to dcm + host. CONCLUSIONS: ecoR II M gene closely linking to ecoR II R gene was very important for the existence of the R-M system in process of evolution, but the key to control EcoR II R enzyme acted later than EcoR II M enzyme did not exist in transcriptional level.

Cloning, Molecular↗

PubMed2000

Closed reduction and percutaneous K-wires fixation of displaced supracondylar humerus fractures in children.

OBJECTIVE: To observe the effects of closed reduction and percutaneous K-wires fixation of displaced supracondylar humerus fracture in children. METHODS: Retrospective review of fourteen patients who sustained displaced supracondylar fracture of distal humerus treated by closed reduction and percutaneous K-wires fixation. RESULTS: All patients' K-wires were removed at 4 weeks post-operation. Their elbow function regained at weeks. The average period of followed up was 10 month (varies from 6 to 18 month), all fractures healed without any permanent complications. Two transient nerves palsy, ulnar and radial nerve each, recovered 12 weeks and 16 weeks post-operation respectively. CONCLUSION: Closed reduction and percutaneous K-wires fixation is a safe and efficient treatment for humerus surpracondylar fracture in children.

Adolescent↗

PubMed2000

The treatment of lumbosacral instability by the Diapason system.

OBJECTIVE: To introduce a new internal fixation system of spine and its characteristics. METHODS: To review 16 patients with lumbosacral instability who were their clinical outcomes and radiographic evaluation. RESULTS: Fifteen patients gained complete recovery from their preoperative symptoms. One patient who had experienced two operations before and with problems of urinary and fecal incontinence and walking difficulty still had lower limb pain, muscle weakness and urinary incontinence after operation. There is no evidence of spine glide on X-ray, implant failure, neural complication or infection during follow up. CONCLUSION: Diapason system can achieve good early postoperative results with few complications and ease to use for lumbosacral instability.

Bone Screws↗

PubMed2000

[Clinical observation of semiconductor laster for treatment of hypersensitivity of the dentine]

OBJECTIVE: Observation of clinical results and SEM features in treatment of hypersensitive dentine by semiconductor laser.METHODS:Semiconductor laser was compared with conventional NaF method for the treatment of 100 teeth with typical hypersensitive dentine which were divided into two groups.RESULTS:The efficiency of the laser group was 96%,while that of NaF group was 76%.The laser irradiation could decrease hypersensitive dentive. There was marked difference between two groups.It was testified by SEM that the dentinal tubule was sealed by semiconductor laser irradiation.CONCLUSION:The safe,effective and easy methods was provided for treating the hypersentitive dentive by use of semiconductor laser.

Journal Article↗

PubMed1999

Increased p27Kip1 cyclin-dependent kinase inhibitor gene expression following anti-IgM treatment promotes apoptosis of WEHI 231 B cells.

Engagement of the B cell receptor of WEHI 231 immature B cells leads sequentially to a drop in c-Myc, to induction of the cyclin-dependent kinase inhibitor p27Kip1, and finally to apoptosis. Recently we demonstrated that the drop in c-Myc expression promotes cell death, whereas the induction of p27 has been shown to lead to growth arrest. In this paper, we demonstrate that increased p27 expression also promotes apoptosis of WEHI 231 B cells. The rescue of WEHI 231 cells by CD40 ligand engagement of its receptor prevented the increase in p27 induction. Inhibition of p27-ablated apoptosis induced upon expression of antisense c-myc RNA. Furthermore, specific induction of p27 gene expression resulted in apoptosis of WEHI 231 cells. Lastly, inhibition of expression of c-Myc, upon induction of an antisense c-myc RNA vector, was sufficient to induce increased p27 levels and apoptosis. Thus, these findings define a signaling pathway during B cell receptor engagement in which the drop in c-Myc levels leads to an increase in p27 levels that promotes apoptosis.

Animals↗

PubMed1999

In vitro and in vivo antitumor activity of a novel immunomodulatory drug, leflunomide: mechanisms of action.

Leflunomide, a novel immunomodulatory drug, has two biochemical activities: inhibition of tyrosine phosphorylation and inhibition of pyrimidine nucleotide synthesis. In the present study, we first showed that A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], the active metabolite of leflunomide, was more effective at inhibiting the tyrosine kinase activity of platelet-derived growth factor (PDGF) receptor than that of epidermal growth factor (EGF) receptor, and had no effect on the tyrosine kinase activity of the fibroblast growth factor receptor. In the presence of exogenous uridine, A77 1726 was more effective at inhibiting the PDGF-stimulated proliferation of PDGF receptor-overexpressing C6 glioma than the EGF-stimulated proliferation of EGF receptor-overexpressing A431 cells. In vivo studies demonstrated that leflunomide treatment strongly inhibited the growth of the C6 glioma but had only a modest effect on the growth of the A431 tumor. Uridine co-administered with leflunomide did not reverse the antitumor activity of leflunomide on C6 and A431 tumors significantly. Quantitation of nucleotide levels in the tumor tissue revealed that leflunomide treatment significantly reduced pyrimidine nucleotide levels in the fast-growing C6 glioma but had no effect on the relatively slow-growing A431 tumor. Whereas uridine co-administration normalized pyrimidine nucleotide levels, it had minimal effects on the antitumor activity of leflunomide in both tumor models. Immunohistochemical analysis revealed that leflunomide treatment significantly reduced the number of proliferating cell nuclear antigen-positive cells in C6 glioma, and that uridine only partially reversed this inhibition. These results collectively suggest that the in vivo antitumor effect of leflunomide is largely independent of its inhibitory effect on pyrimidine nucleotide synthesis. The possibility that leflunomide exerts its antitumor activity by inhibition of tyrosine phosphorylation or by a yet unidentified mode of action is discussed.

3T3 Cells↗

PubMed1999

[Biological characteristics of human fetal esophageal epithelial cell line immortalized by the E6 and E7 gene of HPV type 18].

OBJECTIVE: Biological features including proliferation, differentiation and cell death of SHEE cell line, an immortalized epithelium of the fetal esophageal epithelium induced by HPV 18 E6E7 AAV, were studied. METHODS: SHEE cell line being cultured for more than 50 passages, were cultured in 199 growth medium and were examined by light, electron and fluorescence microscopy for growth rate, morphological features and chromosome analysis, by flow cytometry for cell proliferative dynamics, by immunohistochemistry for Ki67 and cytokeratin, and by terminal DNA transferase label (TUNEL) for apoptosis. RESULTS: At the 20th passage, the SHEE cell remained monolayer, anchorage-dependent and attachment-inhibited growth. The growth curve showed proliferative phase 3-8th days, top-plate phase 9-10th days and attenuative phase after 10th days. Proliferative index(PIx) 34.0%, mitotic index (MI) 2.74% (1.20%-4.80%), apoptotic index (AI) 1.30%-6.90%, chromosome analysis mainly 46 (44-54/nucleus) and DNA distribution in diploidy were calculated and described. The tonofilament expression in cell cytoplasm by electron-microscopy and positive reaction of cytokeratin by immunochemistry showed differentiative character of squamous epithelium. The cell apoptosis occured in the proliferative phase and especially increased in attenuative phase. CONCLUSION: Of biological behaviors, the SHEE cells are close to the basal cells of their original fetal esophageal mucosa keeping proliferative and differentiative potency. This study suggests that the cell death (including cell apoptosis) may be an important factor in studying of cell growth regulation and it may be an research area for cellular biological behaviors.

Cell Division↗

PubMed1999

Integrin family of cell adhesion molecules in the injured brain: regulation and cellular localization in the normal and regenerating mouse facial motor nucleus.

Integrins are a large family of heterodimeric glycoproteins that play a crucial role in cell adhesion during development, inflammation, and tissue repair. In the current study, we investigated the localization of different integrin subunits in the mouse facial motor nucleus and their regulation after transection of the facial nerve. In the normal mouse brain, there was clear immunoreactivity for alpha5-, alpha6-, and beta1-integrin subunits on blood vessel endothelia and for alphaM- and beta2-subunits on resting parenchymal microglia. Facial nerve transection led to an up-regulation of the beta1-subunit on the axotomized neurons and an increase in the alpha4-, alpha5-, alpha6-, beta1-, alphaM-, alphaX-, and beta2-subunits on the adjacent, activated microglia. Quantification of the microglial integrins revealed two different expression patterns. The subunits alpha5 and alpha6 showed a monophasic increase with a maximum at day 4, the alphaM-subunit a biphasic regulation, with an early peak at day 1 and an elevated plateau between day 14 and 42. At day 14, there was also an influx of lymphocytes immunoreactive for the alpha4beta1- and alphaLbeta2-integrins, which aggregated at sites of neural debris and phagocytotic microglia. This finding was accompanied by a significant increase of the alpha5beta1-integrin on blood vessel endothelia. In summary, facial axotomy is followed by a strong and cell-type-specific expression of integrins on the affected neurons and on surrounding microglia, lymphocytes, and vascular endothelia. The presence of several, strikingly different temporal patterns suggests a selective involvement of these molecules in the different adhesive events during regeneration in the central nervous system.

Animals↗

PubMed1999

In vivo nuclear magnetic resonance spectroscopy studies of the relationship between the glutamate-glutamine neurotransmitter cycle and functional neuroenergetics.

In this article we review recent studies, primarily from our laboratory, using 13C NMR (nuclear magnetic resonance) to non-invasively measure the rate of the glutamate-glutamine neurotransmitter cycle in the cortex of rats and humans. In the glutamate-glutamine cycle, glutamate released from nerve terminals is taken up by surrounding glial cells and returned to the nerve terminals as glutamine. 13C NMR studies have shown that the rate of the glutamate-glutamine cycle is extremely high in both the rat and human cortex, and that it increases with brain activity in an approximately 1:1 molar ratio with oxidative glucose metabolism. The measured ratio, in combination with proposals based on isolated cell studies by P. J. Magistretti and co-workers, has led to the development of a model in which the majority of brain glucose oxidation is mechanistically coupled to the glutamate-glutamine cycle. This model provides the first testable mechanistic relationship between cortical glucose metabolism and a specific neuronal activity. We review here the experimental evidence for this model as well as implications for blood oxygenation level dependent magnetic resonance imaging and positron emission tomography functional imaging studies of brain function.

Ammonia↗

PubMed1999

Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with minK subunits.

Long QT syndrome is an inherited disorder of cardiac repolarization caused by mutations in cardiac ion channel genes, including KVLQT1. In this study, the functional consequences of three long QT-associated missense mutations in KvLQT1 (R243C, W248R, E261K) were characterized using the Xenopus oocyte heterologous expression system and two-microelectrode voltage clamp techniques. These mutations are located in or near the intracellular linker between the S4 and S5 transmembrane domains, a region implicated in activation gating of potassium channels. The E261K mutation caused loss of function and did not interact with wild-type KvLQT1 subunits. R243C or W248R KvLQT1 subunits formed functional channels, but compared with wild-type KvLQT1 current, the rate of activation was slower, and the voltage dependence of activation and inactivation was shifted to more positive potentials. Co expression of minK and KvLQT1 channel subunits induces a slow delayed rectifier K(+) current, I(Ks), characterized by slow activation and a markedly increased magnitude compared with current induced by KvLQT1 subunits alone. Coexpression of minK with R243C or W248R KvLQT1 subunits suppressed current, suggesting that coassembly of mutant subunits with minK prevented normal channel gating. The decrease in I(Ks) caused by loss of function or altered gating properties explains the prolonged QT interval and increased risk of arrhythmia and sudden death associated with these mutations in KVLQT1.

Animals↗

PubMed1999

Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes.

BACKGROUND: Insulin resistance, a major factor in the pathogenesis of type 2 diabetes mellitus, is due mostly to decreased stimulation of glycogen synthesis in muscle by insulin. The primary rate-controlling step responsible for the decrease in muscle glycogen synthesis is not known, although hexokinase activity and glucose transport have been implicated. METHODS: We used a novel nuclear magnetic resonance approach with carbon-13 and phosphorus-31 to measure intramuscular glucose, glucose-6-phosphate, and glycogen concentrations under hyperglycemic conditions (plasma glucose concentration, approximately 180 mg per deciliter [10 mmol per liter]) and hyperinsulinemic conditions in six patients with type 2 diabetes and seven normal subjects. In vivo microdialysis of muscle tissue was used to determine the gradient between plasma and interstitial-fluid glucose concentrations, and open-flow microperfusion was used to determine the concentrations of insulin in interstitial fluid. RESULTS: The time course and concentration of insulin in interstitial fluid were similar in the patients with diabetes and the normal subjects. The rates of whole-body glucose metabolism and muscle glycogen synthesis and the glucose-6-phosphate concentrations in muscle were approximately 80 percent lower in the patients with diabetes than in the normal subjects under conditions of matched plasma insulin concentrations. The mean (+/-SD) intracellular glucose concentration was 2.0+/-8.2 mg per deciliter (0.11+/-0.46 mmol per liter) in the normal subjects. In the patients with diabetes, the intracellular glucose concentration was 4.3+/-4.9 mg per deciliter (0.24+/-0.27 mmol per liter), a value that was 1/25 of what it would be if hexokinase were the rate-controlling enzyme in glucose metabolism. CONCLUSIONS: Impaired insulin-stimulated glucose transport is responsible for the reduced rate of insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes mellitus.

Adult↗

PubMed1999

In vivo activity of leflunomide: pharmacokinetic analyses and mechanism of immunosuppression.

BACKGROUND: Leflunomide is an experimental drug with demonstrated ability to prevent and reverse acute allograft and xenograft rejection. The two biochemical activities reported for the active metabolite of leflunomide, A77 1726, are inhibition of tyrosine phosphorylation and inhibition of dihydroorotate dehydrogenase, an enzyme necessary for de novo pyrimidine synthesis. These activities can be distinctly separated in vitro by the use of uridine, which reverses the anti-proliferative effects of A77 1726 caused by inhibition of de novo pyrimidine synthesis. We report the effect of uridine on the in vivo immunosuppressive activities of leflunomide. METHODS: We first quantified the serum levels of A77 1726, the active metabolite of leflunomide, after a single treatment of leflunomide (5, 15, and 35 mg/kg). Additionally, we quantified the levels of serum uridine and of nucleotide triphosphates in the liver, spleen, and lymph nodes of Lewis rats after the administration of a single dose of uridine (500 mg/kg; i.p.). Lewis rats heterotopically transplanted with brown Norway or Golden Syrian hamster hearts were treated for 50 or 75 days with leflunomide (5, 15, and 35 mg/kg/day; gavage) alone or in combination with uridine (500 mg/ kg/day; i.p.). Hematocrits were determined and the levels of alloreactive or xenoreactive immunoglobulin (Ig)M and IgG were determined by flow cytometric analysis. The allograft and xenografts, small bowel, liver, kidney, and spleen were subjected to pathological examination. RESULTS: A linear relationship was observed between the serum A77 1726 concentrations in Lewis rats and the dose of leflunomide administered. Peak A77 1726 concentrations were 20.9, 71.8 and 129.3 mg/l (77.5, 266.1 and 478.8 microM) for the 5, 15, and 35 mg/kg doses of leflunomide, respectively. The concentration of uridine in the serum of normal Lewis rats is 6.5 microM; after i.p. administration of 500 mg/kg uridine, the serum uridine concentrations peaked at 384.1 microM in 15-30 min. The rapid elimination of uridine was not reflected in the lymphoid compartments, and the pharmacokinetics of pyrimidine nucleotides in the spleen resembled that of A77 1726. This dose of uridine, when administered daily (500 mg/kg/day, i.p.), weakly antagonized the immunosuppressive activities of leflunomide (5, 15, and 35 mg/kg/day) in the allotransplantation model. In contrast, in the xenotransplantation model, the same concentration of uridine completely antagonized the immunosuppressive activities of low-dose leflunomide (15 mg/kg/day) and partially antagonized the immunosuppressive activities of high-dose leflunomide (35 mg/kg/day). Toxicities associated with high-dose leflunomide (35 mg/kg/day) were anemia, diarrhea, and pathological changes in the small bowel and liver. These toxicities were significantly reduced by uridine co-administration. CONCLUSION: These studies reveal that the blood levels of A77 1726 in Lewis rats satisfy in vitro requirements for both inhibition of de novo pyrimidine synthesis and protein tyrosine kinase activity. Our data also illustrate that the in vivo mechanism of immunosuppression by leflunomide is complex and is affected by at least the following four factors: type and vigor of the immune response, availability of uridine for salvage by proliferating lymphocytes, species being investigated, and concentration of serum A77 1726.

Aniline Compounds↗

PubMed1999

Determination of the rate of the glutamate/glutamine cycle in the human brain by in vivo 13C NMR.

Recent 13C NMR studies in rat models have shown that the glutamate/glutamine cycle is highly active in the cerebral cortex and is coupled to incremental glucose oxidation in an approximately 1:1 stoichiometry. To determine whether a high level of glutamatergic activity is present in human cortex, the rates of the tricarboxylic acid cycle, glutamine synthesis, and the glutamate/glutamine cycle were determined in the human occipital/parietal lobe at rest. During an infusion of [1-13C]-glucose, in vivo 13C NMR spectra were obtained of the time courses of label incorporation into [4-13C]-glutamate and [4-13C]-glutamine. Using a metabolic model we have validated in the rat, we calculated a total tricarboxylic acid cycle rate of 0.77 +/- 0.07 micromol/min/g (mean +/- SD, n = 6), a glucose oxidation rate of 0.39 +/- 0.04 micromol/min/g, and a glutamate/glutamine cycle rate of 0.32 +/- 0.05 micromol/min/g (mean +/- SD, n = 6). In agreement with studies in rat cerebral cortex, the glutamate/glutamine cycle is a major metabolic flux in the resting human brain with a rate approximately 80% of glucose oxidation.

Adult↗

PubMed1999

Points of Zero Charge and Intrinsic Equilibrium Constants of Silica-Magnetite Composite Oxides.

A series of magnetic adsorbents (silica-magnetite composite oxides) containing 0, 20, 40, 60, 80, and 100 wt% magnetite were prepared via the sol-gel process, where 0.1-µm-radius magnetite particles were encased in a mesoporous silica matrix. The experimental pHpzc for each of the pure oxides and their mixtures was tested against two applicable models in the literature; neither model explained the observed behavior. One of the models did not account for the heterogeneous nature of the two distinct surfaces present in these silica-magnetite composite oxides, and neither model accounted for the possibility of the silica locally altering the behavior of the magnetite functional groups. The behavior was corroborated independently, however, based on an analysis carried out with the measured surface acidity constants. This work also showed that the total exchange capacity of each of the mixed oxides was dominated by the high surface area of the silica, and that the electrolyte species, Na+ or NO-3, did not completely complex with the surface of the oxides, as expected for weak binding ions. The chemical component of the free energy of adsorption of protons for the acid-base reactions also dominated the solvation and coulombic contributions for each of the pure oxides and their mixtures. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

The Alzheimer-related gene presenilin 1 facilitates notch 1 in primary mammalian neurons.

The normal functional neurobiology of the Alzheimer's disease (AD) related gene presenilin 1 (PS1) is unknown. One clue comes from a genetic screen of Caenorhabditis elegans, which reveals that the presenilin homologue sel-12 facilitates lin-12 function [D. Levitan, I. Greenwald, Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene, Nature 377 (1995) 351-355]. The mammalian homologue of lin-12, Notch1, is a transmembrane receptor that plays an important role in cell fate decisions during development, including neurogenesis, but does not have a known function in fully differentiated cells. To better understand the potential role of Notch1 in mammalian postmitotic neurons and to test the hypothesis that Notch and PS 1 interact, we studied the effect of Notch1 transfection on neurite outgrowth in primary cultures of hippocampal/cortical neurons. We demonstrate that Notch1 inhibits neurite extension, and thus has a function in postmitotic mature neurons in the mammalian CNS. Furthermore, we present evidence demonstrating that there is a functional interaction between PS1 and Notch1 in mammalian neurons, analogous to the sel-12/lin-12 interaction in vulval development in C. elegans [D. Levitan, T. Doyle, D. Brousseau, M. Lee, G. Thinakaran, H. Slunt, S. Sisodia, I. Greenwald, Assessment of normal and mutant human presenilin function in Caenorhabditis elegans, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 14940-14944; D. Levitan, I. Greenwald, Effect of Sel-12 presenilin on Lin-12 localization and function in Caenorhabditis elegans, Development, 125 (1998) 3599-3606]. The inhibitory effect of Notch1 on neurite outgrowth is markedly attenuated in neurons from PS1 knockout mice, and enhanced in neurons from transgenic mice overexpressing wild type PS1, but not mutant PS1. These data suggest that PS1 facilitates Notch1 function in mammalian neurons, and support the hypothesis that a functional interaction exists between PS1 and Notch1 in postmitotic mammalian neurons.

Alzheimer Disease↗

PubMed1999

Proteolytic release and nuclear translocation of Notch-1 are induced by presenilin-1 and impaired by pathogenic presenilin-1 mutations.

The Notch family of proteins consists of transmembrane receptors that play a critical role in the determination of cell fate. Genetic studies in Caenorhabditis elegans suggest that the presenilin proteins, which are associated with familial Alzheimer's disease, regulate Notch signaling. Here we show that proteolytic release of the Notch-1 intracellular domain (NICD), an essential step in the activation of Notch signaling, is markedly reduced in presenilin-1 (PS1)-deficient cells and is restored by PS1 expression. Nuclear translocation of the NICD is also markedly reduced in PS1-deficient cells, resulting in reduced transcriptional activation. Mutations in PS1 that are associated with familial Alzheimer's disease impair the ability of PS1 to induce proteolytic release of the NICD and nuclear translocation of the cleaved protein. These results suggest that PS1 plays a central role in the proteolytic activation of the Notch-1-signaling pathway and that this function is impaired by pathogenic PS1 mutations. Thus, dysregulation of proteolytic function may underlie the mechanism by which presenilin mutations cause Alzheimer's disease.

Alzheimer Disease↗

PubMed1999

Pathways of As(III) detoxification in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has two independent transport systems for the removal of arsenite from the cytosol. Acr3p is a plasma membrane transporter that confers resistance to arsenite, presumably by arsenite extrusion from the cells. Ycf1p, a member of the ABC transporter superfamily, catalyzes the ATP-driven uptake of As(III) into the vacuole, also producing resistance to arsenite. Vacuolar accumulation requires a reductant such as glutathione, suggesting that the substrate is the glutathione conjugate, As(GS)3. Disruption of either the ACR3 or YCF1 gene results in sensitivity to arsenite and disruption of both genes produces additive hypersensitivity. Thus, Acr3p and Ycf1p represent separate pathways for the detoxification of arsenite in yeast.

ATP-Binding Cassette Transporters↗

PubMed1999

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts only postnatally. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

Amino Acid Sequence↗