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L D Quarles

Publications and source records attributed to L D Quarles.

At least 19 recordsLinked to original sources
PubMed2004

Selective deficiency of the "bone-related" Runx2-II unexpectedly preserves osteoblast-mediated skeletogenesis.

Runx2 (runt-related transcription factor 2) is a master regulator of skeletogenesis. Distinct promoters in the Runx2 gene transcribe the "bone-related" Runx2-II and non-osseous Runx2-I isoforms that differ only in their respective N termini. Existing mutant mouse models with both isoforms deleted exhibit an arrest of osteoblast and chondrocyte maturation and the complete absence of mineralized bone, but they do not distinguish the separate functions of the two N-terminal isoforms. To elucidate the function of the bone-related isoform, we generated selective Runx2-II-deficient mice by the targeted deletion of the distal promoter and exon 1. Homozygous Runx2-II-deficient (Runx2-II(-/-)) mice unexpectedly formed axial, appendicular, and craniofacial bones derived from either intramembranous ossification or mesenchymal cells of the bone collar, but they failed to form the posterior cranium and other bones derived from endochondral ossification. Heterozygous Runx2-II-deficient mice had grossly normal skeletons, but were osteopenic. The commitment of mesenchymal cells ex vivo to the osteoblast lineage occurred in Runx2-II(-/-) mice, but osteoblastic gene expression was impaired. Chondrocyte maturation appeared normal, but the zone of hypertrophic chondrocytes was not transformed into metaphyseal bone, leading to widened growth plates in Runx2-II(-/-) mice. Compensatory increments in Runx2-I expression occurred in Runx2-II(-/-) mice but were not sufficient to normalize osteoblastic maturation or transcriptional activity. Our findings support distinct functions of Runx2-II and -I in the control of skeletogenesis. Runx2-I is sufficient for early osteoblastogenesis and intramembranous bone formation, whereas Runx2-II is necessary for complete osteoblastic maturation and endochondral bone formation.

Alkaline Phosphatase↗
PubMed2004

MEPE has the properties of an osteoblastic phosphatonin and minhibin.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed exclusively in osteoblasts, osteocytes and odontoblasts with markedly elevated expression found in X-linked hypophosphatemic rickets (Hyp) osteoblasts and in oncogenic hypophosphatemic osteomalacia (OHO) tumors. Because these syndromes are associated with abnormalities in mineralization and renal phosphate excretion, we examined the effects of insect-expressed full-length human-MEPE (Hu-MEPE) on serum and urinary phosphate in vivo, (33)PO(4) uptake in renal proximal tubule cultures and mineralization of osteoblast cultures. Dose-dependent hypophosphatemia and hyperphosphaturia occurred in mice following intraperitoneal (IP) administration of Hu-MEPE (up to 400 microg kg(-1) 31 h(-1)), similar to mice given the phosphaturic hormone PTH (80 microg kg(-1) 31 h(-1)). Also the fractional excretion of phosphate (FEP) was stimulated by MEPE [65.0% (P < 0.001)] and PTH groups [53.3% (P < 0.001)] relative to the vehicle group [28.7% (SEM 3.97)]. In addition, Hu-MEPE significantly inhibited (33)PO(4) uptake in primary human proximal tubule renal cells (RPTEC) and a human renal cell line (Hu-CL8) in vitro (V(max) 53.4% inhibition; K(m) 27.4 ng/ml, and V(max) 9.1% inhibition; K(m) 23.8 ng/ml, respectively). Moreover, Hu-MEPE dose dependently (50-800 ng/ml) inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Inhibition of mineralization was localized to a small (2 kDa) cathepsin B released carboxy-terminal MEPE peptide (protease-resistant) containing the acidic serine-aspartate-rich motif (ASARM peptide). We conclude that MEPE promotes renal phosphate excretion and modulates mineralization.

Amino Acid Sequence↗
PubMed2001

Characterization of the upstream mouse Cbfa1/Runx2 promoter.

Cbfa1 (or Runx2/AML-3/PEPB2alpha) is a transcriptional activator of osteoblastic differentiation. To investigate the regulation of Cbfa1 expression, we isolated and characterized a portion of the 5'-flanking region of the Cbfa1 gene containing its "bone-related" or P1 promoter and exon 1. We identified additional coding sequence in exon 1 and splice donor sites that potentially give rise to a novel Cbfa1 isoform containing an 18 amino acid insert. In addition, primer extension mapping identified in the Cbfa1 promoter a minor mRNA start site located approximately 0.8 kb 5' upstream of the ATG encoding the MASN/p57 isoform and approximately 0.4 kb upstream of the previously reported start site. A luciferase reporter construct containing 1.4 kb of the mouse Cbfa1 promoter was analyzed in Ros 17/2.8 and MC3T3-E1 osteoblast cell lines that express high levels of Cbfa1 transcripts. The activity of this construct was also examined in non-osteoblastic Cos-7 and NIH3T3 cells that do not express Cbfa1 and mesenchymal-derived cell lines, including CH3T101/2, C2C12, and L929 cells, that express low levels of mature Cbfa1 transcripts. The 1.4 kb 5' flanking sequence of the Cbfa1 gene directed high levels of transcriptional activity in Ros 17/2.8 and MC3T3-E1 osteoblasts compared to non-osteoblasts Cos-7 cells, but this construct also exhibited high levels of expression in C310T1/2, L929, and C2C12 cells as well as NIH3T3 cells. In addition, Cbfa1 mRNA expression, but not the activity of the Cbfa1 promoter, was upregulated in a dose-dependent manner in pluripotent mesenchymal C2C12 by bone morphogenetic protein-2 (BMP-2). These data indicate that Cbfa1 is expressed in osteogenic as well as non-osteogenic cells and that the regulation of Cbfa1 expression is complex, possibly involving both transcriptional and post-transcriptional mechanisms. Additional studies are needed to further characterize important regulatory elements and to identify additional regions of the promoter and/or post-transcriptional events responsible for the cell-type restricted regulation of Cbfa1 expression.

3T3 Cells↗
PubMed2001

Effect of asymmetric dimethylarginine on osteoblastic differentiation.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) that accumulates in renal insufficiency and may be a uremic toxin. To determine whether ADMA inhibits bone metabolism, we investigated the in vitro effect of ADMA on osteoblastic differentiation in mouse bone marrow-derived mesenchymal stem cells (BMSCs). METHODS: The effect of ADMA on nitric oxide (NO) production was determined by measuring the stable end product of NO, nitrite, in the culture medium using commercial NO kit. The temporal sequence of osteoblastic differentiation in BMSCs was assessed in the presence and absence of ADMA by measuring alkaline phosphatase (ALP) activity, mineralization, and osteoblast gene expression at 0, 4, 8, 12 days of culture. RESULTS: ADMA (5, 50, 500 micromol. L-1) resulted in a dose-dependent decrease in nitrite formation in conditioned media of BMCS cultures, consistent with inhibition of NOS. ADMA treatment was associated with reduced ALP activity, calcium deposition and osteoblast-related gene expression in BMSCs cultures. Concurrent treatment with l-arginine (3600 micromol. L-1) reversed the ADMA (500 micromol. L-1)-mediated decrease in NO production, restored the differentiation potential of BMSCs, and significantly attenuated the down-regulation of Cbfa1 and osteocalcin gene expression by ADMA. CONCLUSIONS: ADMA inhibition of the NO-NOS pathway in BMSCs impairs osteoblastic differentiation of mouse BMSC cultures. These studies further support a role of NO in the local regulation of bone metabolism and the possibility that ADMA may act as uremic toxin on bone through its effect to inhibit NO actions in osteoblasts.

Alkaline Phosphatase↗
PubMed2001

Analysis of recombinant Phex: an endopeptidase in search of a substrate.

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3'M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3'M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

Amino Acid Sequence↗
PubMed2001

Rickets in cation-sensing receptor-deficient mice: an unexpected skeletal phenotype.

The hypothesis that local changes in extracellular calcium may serve a physiological role in regulating osteoblast, osteoclast, and cartilage function through the extracellular cation-sensing receptor, CasR, is gaining widespread support, but lacks definite proof. To examine the effects of CasR deficiency on the skeleton, we performed a detailed analysis of the skeleton in CasR knockout mice (CasR(-/-)) and wild-type littermates (CasR(+/+)). CasR ablation in the parathyroid glands of CasR(-/-) mice resulted in hyperparathyroidism, hypercalcemia, and hypophosphatemia. Except for dwarfism, the expected skeletal manifestations of PTH excess, namely chondrodysplasia and increased mineralized bone formation and resorption, were not the main skeletal features in CasR(-/-) mice. Rather, rickets was the predominant skeletal abnormality in these animals, as evidenced by a widened zone of hypertrophic chondrocytes, impaired growth plate calcification and disorderly deposition of mineral, excessive osteoid accumulation, and prolonged mineralization lag time in metaphyseal bone. CasR transcripts were identified in cartilage and bone marrow of CasR(+/+) mice, but not in mineralized bone containing mature osteoblasts and osteocytes. These findings indicate that a calcium-sensing receptor is present in the skeleton, and its absence results in defective mineralization of cartilage and bone by mechanisms that remain to be elucidated.

Animals↗
PubMed2001

Cloning and characterization of the proximal murine Phex promoter.

Phex is an endopetidase that regulates systemic phosphate homeostasis. We investigated Phex gene transcription by cloning and performing functional analysis of the 2736 bp of the 5' flanking region of the mouse Phex gene containing its promoter. We identified a transcription start site, a consensus TATA-box, and multiple potential cis-acting regulator elements. To determine whether the promoter directs cell-type restricted Phex expression, we transfected full-length and 5'-deleted Phex luciferase reporter constructs into various cell lines. Phex-expressing C5.18 chondrocytes displayed the highest activity of the transfected Phex promoter constructs compared with non-Phex-expressing COS-7 cells, whereas promoter activity was intermediate in ROS 17/2.8 osteoblasts and maturation stage-dependent in MC3T3-E1 osteoblasts. Analysis of sequential 5'-deletion mutants of the Phex promoter in ROS 17/2.8 cells revealed bimodal activity, suggesting that both positive and negative cis-acting regions may be present. The chondrogenic factor SOX9 markedly stimulated Phex promoter activity, whereas Cbfa1, PTH, and 1,25(OH)(2)D(3) had no effect. Our findings are consistent with the predominant expression of Phex in bone and cartilage. Additional studies will be needed to confirm the regulatory regions in the Phex promoter that function in a cell-restricted manner.

5' Untranslated Regions↗
PubMed2000

Sensing of extracellular cations in CasR-deficient osteoblasts. Evidence for a novel cation-sensing mechanism.

We isolated osteoblastic cell lines from wild-type (CasR(+/+)) and receptor null (CasR(-/-)) mice to investigate whether CasR is present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from both CasR(+/+) and CasR(-/-) mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasR agonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR(-/-) osteoblasts. The functional responses to these cations were identical in CasR(+/+) and CasR(-/-) osteoblasts. Thus, the absence of CasR alters neither the maturational profile of isolated osteoblast cultures nor their in vitro responses to extracellular cations. In addition, CasR transcripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR(+/+) or CasR(-/-) osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative for CasR protein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts from CasR(-/-) mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism.

Amino Acid Sequence↗
PubMed2000

Comparison of treatments for mild secondary hyperparathyroidism in hemodialysis patients. Durham Renal Osteodystrophy Study Group.

UNLABELLED: Comparison of treatments for mild secondary hyperparathyroidism in hemodialysis patients. BACKGROUND: In the management of patients with mild secondary hyperparathyroidism, it is not known whether calcium supplementation alone is sufficient to correct abnormalities in bone and mineral metabolism or if calcitriol is needed in either physiologic oral or intravenous pharmacologic doses. METHODS: This was a 40-week prospective nonmasked trial of 52 patients [parathyroid hormone (PTH) 150 to 600 pg/mL] who were randomized to receive escalating doses of either calcium carbonate (CaCO3) alone (calcium group, N = 11), daily oral calcitriol (oral group, N = 20), or intermittent intravenous calcitriol (IV group, N = 21). The groups were compared with regard to changes in serum intact PTH, serum bone-specific alkaline phosphatase (BAP), incidence of hypercalcemia (>10.5 mg/dL), and hyperphosphatemia (>6.5 mg/dL). RESULTS: PTH levels decreased in all groups (P < 0.01, paired t-test). In the calcium group, PTH (mean +/- SEM) decreased from 325 +/- 46.2 to 160 +/- 44.5 pg/mL. In the oral group, it decreased from 265 +/- 26.4 to 125 +/- 23.7 pg/mL, and in the IV group, it decreased from 240 +/- 27.7 to 65 +/- 10.0 pg/mL. Upon analysis of covariance, controlling for the initial PTH level, we found no differences in the PTH response between the groups (P > 0.10). In contrast, the BAP concentration increased from 20.7 +/- 7.6 to 27.5 +/- 7.0 microg/L in the calcium group (P = 0.17), decreased from 20. 6 +/- 3.9 to 17.8 +/- 4.5 microg/L in the oral group (P = 0.26), and from 19.1 +/- 2.6 to 10.6 +/- 1.1 microg/L in the IV group (P = 0. 007). Serum calcium increased significantly in all groups from 8.4 +/- 0.25 to 9.0 +/- 0.28, 8.5 +/- 0.16 to 9.2 +/- 0.27, and 8.7 +/- 0.16 to 9.4 +/- 0.18 mg/dL in the calcium, oral, and IV groups, respectively (P = NS difference between groups). Serum phosphorus was significantly lower in the calcium group throughout the study (P = 0.02). Hypercalcemic episodes were 2.0 +/- 0.8, 3.0 +/- 0.6, and 3. 4 +/- 0.6 per patient-year (P > 0.10), and hyperphosphatemic episodes were 0.9 +/- 0.56, 4.2 +/- 0.79 and 4.9 +/- 0.84 in the calcium, oral, and IV groups, respectively (P < 0.01). CONCLUSION: In mild secondary hyperparathyroidism, all three strategies are effective. High-dose CaCO3 alone may be sufficient to control PTH with a favorable side-effect profile, but calcitriol appears to have additional suppressive effects on bone that are greater following the intravenous route of administration and may increase the risk of adynamic bone disease.

Alkaline Phosphatase↗
PubMed1999

Cbfa1 isoform overexpression upregulates osteocalcin gene expression in non-osteoblastic and pre-osteoblastic cells.

The mouse Cbfa1 gene potentially encodes several proteins that differ in their N-terminal sequences, including an osteoblast-specific transcription factor, Cbfa1/Osf2, a Cbfa1 isoform (Cbfa1/iso), and the originally described Cbfa1 gene product (Cbfa1/org). Uncertainty exists about the function of these potential isoforms of the Cbfa1 gene. To examine the transactivation potential of different Cbfa1 gene products, we compared the ability of Cbfa1/Osf2, Cbfa1/iso, and Cbfa1/org overexpression to activate an osteocalcin promoter/reporter construct in NIH3T3 fibroblasts, C3H10T1/2 pluripotent cells and MC3T3-E1 pre-osteoblasts. These three cell lines were transiently cotransfected with a 1.3-kb mouse osteocalcin promoter luciferase-fusion construct (p1.3OC-luc) and different amounts of expression vectors containing the respective full-length Cbfa1 isoform cDNAs. Using transfection protocols with lower amounts of expression plasmid DNAs, we found that all three Cbfa1 isoforms stimulated osteocalcin promoter activity in each of the cell types, consistent with the their ability to induce expression of an osteoblast-specific gene both in non-osteoblast cells and in osteoblast cell lines. However, using transfection protocols with higher amounts of expression plasmids containing Cbfa1 cDNAs, we found that the Cbfa1/Osf2 and Cbfa1/org had less transactivating potential compared with Cbfa1/iso. Our studies suggest that the 87-amino acid N-terminus of Cbfa1/Osf2 is not crucial for optimal transactivation, whereas the 19-amino acid N-terminal sequence of Cbfa1/iso augments transcriptional activation only at high doses of the expression plasmid. The physiological significance of these in vitro findings remain to be determined.

3T3 Cells↗
PubMed1999

Linkage of a gene causing familial focal segmental glomerulosclerosis to chromosome 11 and further evidence of genetic heterogeneity.

Focal segmental glomerulosclerosis (FSGS) is a pathological entity characterized by proteinuria, nephrotic syndrome, and the progressive loss of renal function. It is a common cause of end-stage renal disease (ESRD). Recently, familial forms of FSGS have been identified. Two families with autosomal dominant FSGS were evaluated for linkage using 351 genomic microsatellite markers. Linkage, multipoint analysis, and tests for heterogeneity were performed on the subsequent results. In addition, three small families were used for haplotype analysis. Evidence for linkage was found on chromosome 11q21-q22 for the largest family, with a maximum lod score of 9.89. The gene is currently localized to an 18-cM area between flanking markers D11S2002 and D11S1986. The disease in a second family was not linked to this locus or to a previously described locus on chromosome 19q13. There were no shared haplotypes among affected individuals in the three smaller families. Our findings demonstrate that genetic heterogeneity is prevalent in FSGS in that at least three genes cause the FSGS phenotype. Identification of the genes that cause familial FSGS will provide valuable insights into the molecular basis and pathophysiology of FSGS.

Adolescent↗
PubMed1999

Clinical and genetic heterogeneity in familial focal segmental glomerulosclerosis. International Collaborative Group for the Study of Familial Focal Segmental Glomerulosclerosis.

BACKGROUND: Familial forms of focal segmental glomerulosclerosis (FFSGS) that exhibit autosomal dominant or recessive patterns of inheritance have been described. The genetic basis of these hereditary forms of FSGS is unknown. One recent study of a kindred from Oklahoma with an autosomal dominant form of FSGS linked this disease to a region of chromosome 19q. In addition, polymorphisms in a gene in this region on chromosome 19q13 have been linked to congenital nephrotic syndrome of the Finnish type. We have ascertained and characterized a large family with autosomal dominant FFSGS (Duke 6530). METHODS: Families were compared for clinical and genetic heterogeneity. To test for linkage of our family to this portion of chromosome 19, genomic DNA was isolated from 102 family members, and polymerase chain reaction was performed using eight microsatellite markers that spanned the area of interest on chromosome 19. Data were evaluated using two-point linkage analysis, multipoint analysis, and an admixture test. RESULTS: Linkage was excluded at a distance of +/- 5 to 10 CM for all markers tested with two-point log10 of the odds of linkage (LOD) scores and from an approximate 60 CM interval in this area of chromosome 19q via multipoint analysis. CONCLUSIONS: FSGS has been called the "final common pathway" of glomerular injury, as it is a frequent pathological manifestation with diverse etiologies. This diversity likely correlates with the genetic heterogeneity that we have established. Thus, our data demonstrate that there are at least two genes responsible for this disease, and there is genetic as well as clinical heterogeneity in autosomal dominant FSGS.

Adolescent↗
PubMed1999

Aluminum is a weak agonist for the calcium-sensing receptor.

BACKGROUND: Aluminum (Al3+) has diverse biological effects mediated through activation of a putative extracellular cation-sensing receptor. A recently identified calcium-sensing receptor (CaSR), which has been identified in target tissues for Al3+, may transduce some of the biological effects of Al3+. METHODS: To test this possibility, we transfected human embryonic kidney 293 (HEK 293) cells with a cDNA encoding the rat CaSR and evaluated CaSR expression by Western blot analysis and function by measurement of intracellular calcium ([Ca2+]i) levels and inositol monophosphate (IP1) generation following stimulation with Al3+ and a panel of CaSR agonists. RESULTS: The CaSR protein was detected by immunoblot analysis in cells transfected with the CaSR cDNA but not in nontransfected HEK 293 cells. In addition, [Ca2+]i levels and IP1 generation were enhanced in a dose-dependent fashion by additions of the CaSR agonists calcium (Ca2+), magnesium (Mg2+), gadolinium (Gd3+), and neomycin only in cells transfected with CaSR. To determine if Al3+ activated CaSR, we stimulated cells transfected with rat CaSR with 10 microM to 1 mM concentrations of Al3+. Concentrations of Al3+ in the range of 10 microM to 100 microM had no effect on [Ca2+]i levels or IP1 generation. In contrast, 1 mM Al3+ induced small but significant increases in both parameters. Whereas Gd3+ antagonized calcium-mediated activation of CaSR, pretreatment with Al3+ failed to block subsequent activation of rat CaSR by Ca2+, suggesting a distinct mechanism of Al3+ action. CONCLUSION: Al3+ is not a potent agonist for CaSR. Because Al3+ affects a variety of target tissues at micromolar concentrations, it seems unlikely that CaSR mediates these cellular actions of Al3+.

Aluminum↗
PubMed1999

Spectrum of disease in familial focal and segmental glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is the underlying pathologic entity in 5% of adults and 20% of children with end-stage renal disease (ESRD). FSGS is generally considered to be sporadic in origin. METHODS: Recently, we identified 60 families involving 190 individuals with familial FSGS, providing evidence for a subset of families in which a genetic form is segregating. Each family had at least one member with renal biopsy-confirmed FSGS and at least one other member with either renal biopsy-confirmed FSGS or ESRD. RESULTS: Twenty-six families had individuals affected in more than one generation [multigeneration (MG)], and the remaining 34 families had only a single generation (SG) affected. There was equal representation of males and females among affected individuals. Ten percent of MG families were African American, and 52% of SG families were African American. The mean age of presentation was significantly higher in the MG families (32.5 +/- 14.6 years) compared with the SG families (20.1 +/- 12.1 years, P = 0.0001). SG cases had higher levels of proteinuria at presentation (7.0 +/- 5.6 g/24 hr, compared with 3.8 +/- 3.4 g/24 hr, for the MG families, P = 0.002). On renal biopsy, tubulointerstitial damage was more severe in patients in the SG families than in the MG families; however, the level of glomerular damage did not differ between these groups. Fifty percent of the patients had progressed to ESRD by the age of 30 years. Variables measured at presentation that were independently associated with poor renal survival were decreased age, increased serum creatinine, and increased urinary protein excretion. Forty-one patients underwent successful renal transplantation, with a 10-year graft survival rate of 62%. One patient developed clinical and biopsy evidence of recurrence of FSGS in the allograft. CONCLUSION: These data confirm the existence of a non-Alport's form of hereditary glomerulonephritis, which has a morphological pattern of FSGS.

Adult↗
PubMed1999

Coordinated maturational regulation of PHEX and renal phosphate transport inhibitory activity: evidence for the pathophysiological role of PHEX in X-linked hypophosphatemia.

The mechanism by which inactivating mutations of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) cause X-linked hypophosphatemia remains unknown. However, recent reports suggest errant PHEX activity in osteoblasts may fail to inactivate a phosphaturic factor produced by these cells. To test this possibility, we examined coordinated maturational expression of PHEX and production of phosphate transport inhibitory activity in osteoblasts from normal and hyp-mice. We assessed the inhibitory activity in conditioned medium by examining the effects on opossum kidney cell phosphate transport and osteoblast PHEX expression by reverse transcriptase-polymerase chain reaction during a 17-day maturational period. Inhibitory activity increased as a function of osteoblast maturational stage, with no activity after 3 days and persistent activity by 6 days of culture. More significantly, equal phosphate transport inhibitory activity in conditioned medium from normal and hyp-mouse osteoblasts (control 1.90 +/- 0.12, normal 1.48 +/- 0.10, hyp 1.45 +/- 0.04 nmol/mg of protein/minute) was observed at 6 days. However, by 10 days hyp-mouse osteoblasts exhibited greater inhibitory activity than controls, and by 17 days the difference in phosphate transport inhibition maximized (control 2.08 +/- 0.09, normal 1.88 +/- 0.06, hyp 1.58 +/- 0.06 nmol/mg of protein/minute). Concurrently, we observed absent PHEX expression in normal osteoblasts after 3 days, limited production at 6 days, and significant production by day 10 of culture, while hyp-mouse osteoblasts exhibited limited PHEX activity secondary to an inactivating mutation. The data suggest that the presence of inactivating PHEX mutations results in the enhanced renal phosphate transport inhibitory activity exhibited by hyp-mouse osteoblasts.

Animals↗
PubMed1998

Genomic structure and isoform expression of the mouse, rat and human Cbfa1/Osf2 transcription factor.

Although the CBFA1 gene encodes an osteoblast-specific transcription factor that regulates osteoblast differentiation, uncertainty exists about the organization of its 5' end and the relevance of a novel N-terminal sequence identified in the mouse Cbfa1/Osf2 isoform. We found the novel 5' Cbfa1/Osf2 sequence is encoded by a previously unrecognized upstream exon, designated exon -1, which is highly conserved in mouse, rat and human. In addition, two splice donor sites may be utilized to generate Cbfa1/Osf2 cDNAs containing different N-terminal sequences. The first ATG and splice donor site in exon -1 is predicted to transcribe a cDNA containing the unique Osf2 5' sequence, whereas a second donor splice site gives rise to cDNAs that contain sequences encoding an 11 amino acid insert. In the human CBFA1 gene, an additional 2-bp nucleotide insert shifts the reading frame and results in stop codons in the cDNA sequence derived from exon -1. The 5'-most exon of the human CBFA1 gene, therefore, contains the 5' non-coding region rather than a human OSF2 homolog. The absence of a homologous OSF2 coding sequence in the human CBFA1 cDNA suggests that the novel mouse N-terminal Osf2 sequence is not essential for functioning of the CBFA1 gene product. In addition, multiple transcripts derived from a single CBFA1/Cbfa1 gene were detected in osteoblasts by Northern analysis and RT-PCR, including additional Cbfa1/Osf2 isoforms containing deletions of exons 1 and 4. Thus, the alternative use of transcription start sites and splicing leads to the genesis of CBFA1/Cbfa1 isoforms with possible differences in transactivation potentials.

Amino Acid Sequence↗