Zhousheng Xiao

Regulation of Fibroblastic Growth Factor 23 Expression but Not Degradation by PHEX

Inactivating mutations of Phex cause X-linked hypophosphatemia (XLH) by increasing levels of a circulating phosphaturic factor. FGF23 is a candidate for this phosphaturic factor. Elevated serum FGF23 levels correlate with the degree of hypophosphatemia in XLH, suggesting that loss of Phex function in this disorder results in either diminished degradation and/or increased biosynthesis of FGF23. To establish the mechanisms whereby Phex regulates FGF23, we assessed Phex-dependent hydrolysis of recombinant FGF23 in vitro and measured fgf23 message levels in the Hyp mouse homologue of XLH. In COS-7 cells, overexpression of FGF23 resulted in its degradation into N- and C-terminal fragments by an endogenous decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone-sensitive furin-type convertase. Phex-dependent hydrolysis of full-length FGF23 or its N- and C-terminal fragments could not be demonstrated in the presence or absence of decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone in COS-7 cells expressing Phex and FGF23. In a reticulolysate system, apparent cleavage of FGF23 occurred with wild-type Phex, the inactive Phex-3′M mutant, and vector controls, indicating nonspecific metabolism of FGF23 by contaminating enzymes. These findings suggest that FGF23 is not a direct Phex substrate. In contrast, by real-time reverse transcriptase PCR, the levels of fgf23 transcripts were highest in bone, the predominant site of Phex expression. In addition, Hyp mice displayed a bone-restricted increase in fgf23 transcripts in association with inactivating Phex mutations. Increased expression of fgf23 was also observed in Hyp-derived osteoblasts in culture. These findings suggest that Phex, possibly through the actions of unidentified Phex substrates or other downstream effectors, regulates fgf23 expression as part of a potential hormonal axis between bone and kidney that controls systemic phosphate homeostasis and mineralization.

Cilia-like structures and polycystin-1 in osteoblasts/ osteocytes and associated abnormalities in skeletogenesis and Runx2 expression

We examined the osteoblast/osteocyte expression and function of polycystin-1 (PC1), a transmembrane protein that is a component of the polycystin-2 (PC2)-ciliary mechano-sensor complex in renal epithelial cells. We found that MC3T3-E1 osteoblasts and MLO-Y4 osteocytes express transcripts for PC1, PC2, and the ciliary proteins Tg737 and Kif3a. Immunohistochemical analysis detected cilia-like structures in MC3T3-E1 osteoblastic and MLO-Y4 osteocyte-like cell lines as well as primary osteocytes and osteoblasts from calvaria. Pkd1m1Bei mice have inactivating missense mutations of Pkd1 gene that encode PC1. Pkd1m1Bei homozygous mutant mice demonstrated delayed endochondral and intramembranous bone formation, whereas heterozygous Pkd1m1Bei mutant mice had osteopenia caused by reduced osteoblastic function. Heterozygous and homozygous Pkd1m1Bei mutant mice displayed a gene dose-dependent decrease in the expression of Runx2 and osteoblast-related genes. In addition, overexpression of constitutively active PC1 C-terminal constructs in MC3T3-E1 osteoblasts resulted in an increase in Runx2 P1 promoter activity and endogenous Runx2 expression as well as an increase in osteoblast differentiation markers. Conversely, osteoblasts derived from Pkd1m1Bei homozygous mutant mice had significant reductions in endogenous Runx2 expression, osteoblastic markers, and differentiation capacity ex vivo. Co-expression of constitutively active PC1 C-terminal construct into Pkd1m1Bei homozygous osteoblasts was sufficient to normalize Runx2 P1 promoter activity. These findings are consistent with a possible functional role of cilia and PC1 in anabolic signaling in osteoblasts/osteocytes.

Calcitonin gene-related peptide-induced preconditioning protects against ischemia-reperfusion injury in isolated rat hearts.

Our previous work has suggested that the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) can abolish the protective effect of ischemic preconditioning in the isolated rat heart. Therefore we tested the hypothesis that CGRP- or capsaicin-induced preconditioning protects against ischemia-reperfusion injury in the isolated perfused rat heart. Thirty minutes of global ischemia and 30 min of reperfusion caused a significant cardiac contractile dysfunction, ventricular arrhythmia, and an increased release of creatine phosphate kinase. Pretreatment with CGRP or capsaicin, which evokes release of CGRP from cardiac sensory nerves, for 5 min produced a significant improvement of cardiac function, a reduction in the incidence of ventricular arrhythmia, and a decrease in the release of creatine phosphate kinase. However, the cardioprotection provided by CGRP- or capsaicin-induced preconditioning was abolished by CGRP-(8-37) and ruthenium red, respectively. These findings suggest that CGRP- or capsaicin-induced preconditioning protects against ischemic myocardial injury. The present results also suggest that CGRP may be an endogenous myocardial protective substance in the rat.

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. J. Cell. Biochem. 74:596–605, 1999.

Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture.

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow‐derived mesenchymal stem cells (BMSCs). Gen (10−8 ∼ 10−6 M) resulted in a dose‐dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration‐dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose‐dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17β‐estradiol (E2, 10−8 M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10−7 M) or the NOS inhibitor L‐NAME (3 × 10−3 M) diminished the Gen (10−6 M)‐mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H‐[1,2,4]oxadiazolo [4,3,‐a]quinoxalin‐1‐one (ODQ, 10−6 M) selectively blocked the Gen (10−6 M)‐mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen‐induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen‐like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.

Role of Matrix Extracellular Phosphoglycoprotein in the Pathogenesis of X-Linked Hypophosphatemia

X-linked hypophosphatemia (XLH), a disorder characterized by hypophosphatemia, impaired skeletal mineralization, and aberrant regulation of 1, 25(OH)(2)D(3), is caused by inactivating mutations of Phex, which results in the accumulation of putative phosphaturic factors, called phosphatonins. Matrix extracellular phosphoglycoprotein (Mepe) is a proposed candidate for phosphatonin. The authors found that Hyp mice had increased expression of the MEPE and another phosphaturic factor, Fgf23. To establish MEPEs role in the pathogenesis of the XLH, Mepe-deficient mice were back-crossed onto the Hyp mouse homologue of XLH and phenotypes of wild-type, Mepe(-/-), Hyp, and Mepe(-/-)/Hyp mice were examined. Transfer of Mepe deficiency onto the Phex-deficient Hyp mouse background failed to correct hypophosphatemia and aberrant serum 1,25(OH)(2)D(3) levels. Increased Fgf23 levels in Hyp mice were not affected by superimposed Mepe deficiency. In addition, Mepe-deficient Hyp mice retained bone mineralization defects in vivo, characterized by decreased bone mineral density, reduced mineralized trabecular bone volume, lower flexural strength, and histologic evidence of osteomalacia; however, cultures of Hyp-derived bone marrow stromal cells in the absence of Mepe showed improved mineralization and normalization of osteoblast gene expression profiles observed in cells derived from Mepe-null mice. These results demonstrate that MEPE elevation in Hyp mice does not contribute to the hypophosphatemia associated with inactivating Phex mutations and is therefore not phosphatonin.

Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice

We investigated whether polycystin‐1 is a bone mechanosensor. We conditionally deleted Pkd1 in mature osteoblasts/osteocytes by crossing Dmp1‐Cre with Pkd1flox/m1Bei mice, in which the m1Bei allele is nonfunctional. We assessed in wild‐type and Pkd1‐deficient mice the response to mechanical loading in vivo by ulna loading and ex vivo by measuring the response of isolated osteoblasts to fluid shear stress. We found that conditional Pkd1 heterozygotes (Dmp1‐Cre;Pkd1flox/+) and null mice (Pkd1Dmp1‐cKO) exhibited a ~40 and ~90% decrease, respectively, in functional Pkd1 transcripts in bone. Femoral bone mineral density (12 vs. 27%), trabecular bone volume (32 vs. 48%), and cortical thickness (6 vs. 17%) were reduced proportionate to the reduction of Pkd1 gene dose, as were mineral apposition rate (MAR) and expression of Runx2‐II, Osteocalcin, Dmp1, and Phex. Anabolic load‐induced periosteal lamellar MAR (0.58±0.14; Pkd1Dmp1‐cKO vs. 1.68±0.34 μm/d; control) and increases in Cox‐2, c‐Jun, Wnt10b, Axin2, and Runx2‐II gene expression were significantly attenuated in Pkd1Dmp1‐cKO mice compared with controls. Application of fluid shear stress to immortalized osteoblasts from Pkd1null/null and Pkd1m1Bei/m1Bei‐derived osteoblasts failed to elicit the increments in cytosolic calcium observed in wild‐type controls. These data indicate that polycystin‐1 is essential for the anabolic response to skeletal loading in osteoblasts/osteocytes.—Xiao, Z., Dallas, M., Qiu, N., Nicolella, D., Cao, L., Johnson, M., Bonewald, L., Quarles, L. D. Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice. FASEB J. 25, 2418–2432 (2011). www.fasebj.org

Characterization of the upstream mouse Cbfa1/Runx2 promoter.

Cbfa1 (or Runx2/AML‐3/PEPB2α) 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 ∼0.8 kb 5′ upstream of the ATG encoding the MASN/p57 isoform and ∼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. J. Cell. Biochem. 82: 647–659, 2001.

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.

Polycystin-1 Regulates Skeletogenesis through Stimulation of the Osteoblast-specific Transcription Factor RUNX2-II

Polycystin-1 (PC1) may play an important role in skeletogenesis through regulation of the bone-specific transcription factor Runx2-II. In the current study we found that PC1 co-localizes with the calcium channel polycystin-2 (PC2) in primary cilia of MC3T3-E1 osteoblasts. To establish the role of Runx2-II in mediating PC1 effects on bone, we crossed heterozygous Pkd1m1Bei and Runx2-II mice to create double heterozygous mice (Pkd1+/m1Bei/Runx2-II+/-) deficient in both PC1 and Runx2-II. Pkd1+/m1Bei/Runx2-II+/- mice exhibited additive reductions in Runx2-II expression that was associated with impaired endochondral bone development, defective osteoblast-mediated bone formation, and osteopenia. In addition, we found that basal intracellular calcium levels were reduced in homozygous Pkd1m1Bei osteoblasts. In contrast, overexpression of a PC1 C-tail construct increased intracellular calcium and selectively stimulated Runx2-II P1 promoter activity in osteoblasts through a calcium-dependent mechanism. Site-directed mutagenesis of critical amino acids in the coiled-coil domain of PC1 required for coupling to PC2 abolished PC1-mediated Runx2-II P1 promoter activity. Additional promoter analysis mapped the PC1-responsive region to the “osteoblast-specific” enhancer element between -420 and -350 bp that contains NFI and AP-1 binding sites. Chromatin immunoprecipitation assays confirmed the calcium-dependent binding of NFI to this region. These findings indicate that PC1 regulates osteoblast function through intracellular calcium-dependent control of Runx2-II expression. The overall function of the primary cilium-polycystin complex may be to sense and transduce environmental clues into signals regulating osteoblast differentiation and bone development.