Gang-Qing Yao

Nuclear Factor-κB p50 Is Required for Tumor Necrosis Factor-α-Induced Colony-Stimulating Factor-1 Gene Expression in Osteoblasts1

Colony-stimulating factor (CSF)-1 is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. We have reported that osteoblasts express CSF-1 constitutively and that tumor necrosis factor (TNF)-alpha, a potent bone-resorbing agent, increases CSF-1 gene expression by a transcriptional mechanism. In the present study, we report that an NF-kappaB site in the CSF-1 promoter is required for TNF-alpha-induced CSF-1 expression in osteoblasts. As determined by electrophoretic mobility shift assays, antiserum against the NF-kappaB-binding protein, p50, retarded the mobility of the inducible complex, whereas antisera against p52, p65, c-Rel, Rel B, IkappaB alpha, IkappaB gamma, and Bcl-3 had no effect. To further confirm that p50 is necessary for TNF-alpha-induced CSF-1 expression in osteoblasts, CSF-1 messenger RNA expression from untreated and TNF-alpha-treated osteoblasts, prepared from wild-type and p50 knock-out mice, was examined by Northern analysis. CSF-1 messenger RNA was increased by TNF treatment in wild-type mice but not in NF-kappaB p50 knock-out mice. Our findings support the conclusion that the NF-kappaB subunit p50 is critical for TNF-induced CSF-1 expression in osteoblasts.

A Role for Cell-Surface CSF-1 in Osteoblast-mediated Osteoclastogenesis

CSF-1 is required for osteoblast-mediated osteoclast formation. Osteoblasts produce soluble (sCSF-1) and cell-surface forms of CSF-1 (also known as membrane-bound CSF-1, mCSF-1) but their individual contributions to osteoclastogenesis remain unclear. Using glutaraldehyde-fixed primary murine osteoblasts as a source of mCSF-1, osteoblasts from op/op mice as a source for other osteoblast-derived osteoclastogenic factors and murine bone marrow as a source of osteoclast progenitors, osteoclast-like cells (OCL) formation was observed after 7-9 days of co-culture. In contrast, no OCL formation occurred when mCSF-1 expressed by primary murine osteoblasts was blocked by CSF-1 antibody pretreatment or when op/op osteoblasts were substituted for primary murine osteoblasts in the co-culture system. Osteoclast formation was also significantly inhibited when murine primary osteoblasts were pretreated with an antisense phosphorothioate oligonucleotide against mCSF-1. Finally, mCSF-1 and sCSF-1 were synergistic in stimulating OCL formation. These data support the conclusion that mCSF-1 plays an important role in osteoblast-mediated osteoclastogenesis within the bone microenvironment.

Targeted overexpression of the two colony-stimulating factor-1 isoforms in osteoblasts differentially affects bone loss in ovariectomized mice

Colony-stimulating factor-1 (CSF1) is one of two cytokines required for normal osteoclastogenesis. There are two major isoforms of CSF1, the cell-surface or membrane-bound isoform (mCSF1) and soluble CSF1 (sCSF1). Whether these isoforms serve nonredundant functions in bone is unclear. To explore this question, we generated transgenic mice expressing human sCSF1, human mCSF1, or both (s/mCSF1) in osteoblasts using the 2.3-kb rat alphaI-collagen promoter. Bone density determined by peripheral quantitative computed tomography was significantly reduced in mCSF1, sCSF1, and s/mCSF1 transgenic mice compared with wild-type animals. When analyzed by sex, sCSF1, and s/mCSF1, female animals but not mCSF1 female mice were found to have greater bone loss than their male littermates (-20 vs. -9.2%; P<0.05 for sCSF1 and -21.6 vs. -11.2% for s/mCSF1; P<0.01). By breeding CSF1 isoform-selective transgenic mice to an op/op background, mice were generated in which a single CSF1 isoform was the only source of the cytokine (sCSF1op/op and mCSF1op/op). Unlike osteoblast-targeted overexpression of mCSF1, selective transgenic expression of sCSF1 did not completely correct the op/op phenotype in 5-mo-old animals. Interestingly, compared with sham-ovariectomized mice of the same genotype, ovariectomy in sCSF1op/op mice led to a greater loss of spinal bone mineral density (22.1%) than was seen in either mCSF1op/op mice (12.9%) or in wild-type animals (10.9%). Our findings support the conclusion that sCSF1 and mCSF1 serve nonredundant functions in bone and that sCSF1 may play a role in mediating estrogen-deficiency bone loss.

The transcription factor T-box 3 regulates colony-stimulating factor 1-dependent Jun dimerization protein 2 expression and plays an important role in osteoclastogenesis.

Background: Colony-stimulating factor 1 (CSF1) and Jun dimerization protein 2 (JDP2) regulate osteoclastogenesis, but a molecular interaction between the two has not been reported. Results: CSF1 up-regulates JDP2 by inducing T-box 3 (Tbx3) binding to the JDP2 promoter, and suppressing Tbx3 expression impairs osteoclastogenesis. Conclusion: Tbx3 is a target for CSF1 in osteoclasts. Significance: Novel molecular targets for CSF1 in modulating osteoclastogenesis are described. Colony-stimulating factor 1 (CSF1) is known to promote osteoclast progenitor survival, but its roles in osteoclast differentiation and mature osteoclast function are less well understood. In a microarray screen, Jun dimerization protein 2 (JDP2) was identified as significantly induced by CSF1. Recent reports indicate that JDP2 is required for normal osteoclastogenesis and skeletal metabolism. Because there are no reports on the transcriptional regulation of this gene, the DNA sequence from −2612 to +682 bp (relative to the transcription start site) of the JDP2 gene was cloned, and promoter activity was analyzed. The T box-binding element (TBE) between −191 and −141 bp was identified as the cis-element responsible for CSF1-dependent JDP2 expression. Using degenerate PCR, Tbx3 was identified as the major isoform binding the TBE. Overexpression of Tbx3 induced JDP2 promoter activity, whereas suppressing Tbx3 expression substantially attenuated CSF1-induced transcription. Suppressing Tbx3 in osteoclast precursors reduced JDP2 expression and significantly impaired RANKL/CSF1-induced osteoclastogenesis. A MEK1/2-specific inhibitor was found to block CSF1-induced JDP2 expression. Consistent with these data, JDP2−/− mice were found to have increased bone mass. In summary, CSF1 up-regulates JDP2 expression by inducing Tbx3 binding to the JDP2 promoter. The downstream signaling cascade from activated c-Fms involves the MEK1/2-ERK1/2 pathway. Tbx3 plays an important role in osteoclastogenesis at least in part by regulating CSF1-dependent expression of JDP2.

The Cell-Surface Isoform of Colony Stimulating Factor 1 (CSF1) Restores but Does Not Completely Normalize Fecundity in CSF1-Deficient Mice

Abstract The complete genetic absence of colony stimulating factor 1 (CSF1) in CSF1-deficient Csf1op/Csf1op mice leads to reproductive defects in males and females. Although the cell-surface or membrane-bound isoform of CSF1 (mCSF1) is biologically active in bone, little is known about its role in reproduction. Transgenic mice expressing mCSF1 under the control of the 2.4-kb rat collagen type I alpha promoter were developed [Tg(Col1a1-mCSF1)1Gqy] and bred onto a Csf1op/Csf1op background [Csf1op/Csf1op; Tg(Col1a1-mCSF1)1Gqy] to examine the effects of the mCSF1 isoform in bone in vivo. Surprisingly, when interbred, these mice were fertile. The Csf1op/Csf1op; Tg(Col1a1-mCSF1)1Gqy transgenic male mice have normal libido, sperm number and percent of motile sperm. In Csf1op/Csf1op; Tg(Col1a1-mCSF1)1Gqy females, puberty and estrus cycles are at expected age and duration. Further, females are able to carry pregnancies to term and nurse their offspring. Crosses of Csf1op/Csf1op; Tg(Col1a1-mCSF1)1Gqy males or females with their control littermates showed no significant differences in either number or viability of offspring. However, crossing Csf1op/Csf1op; Tg(Col1a1-mCSF1)1Gqy males with Csf1op; Tg(Col1a1-mCSF1)1Gqy females resulted in a decline in both the number and viability of offspring, suggesting that a subtle reproductive defect might persist in the transgenic animals that was only manifest when the animals were interbred. Although the gravid murine uterus expresses extremely high levels of CSF1 that are thought to be important for reproduction, uterine tissue levels of CSF1 remained low and unchanged during pregnancy in Csf1op/Csf1op; Tg(Col1a1-mCSF1)1Gqy mice. Low levels of CSF1 protein were detected in serum and in lung and uterine tissue in Csf1op/Csf1op; Tg(Col1a1-mCSF1)1Gqy mouse, which likely result from the known proteolytic shedding of mCSF1 from the cell surface. These data are consistent with the conclusion that mCSF1, when shed from the cell surface, can support reproduction and that high uterine tissue levels of CSF1 may not be required for mouse reproduction.

CSF-1 Induces fos Gene Transcription and Activates the Transcription Factor Elk-1 in Mature Osteoclasts

Mice with targeted deletion of the fos gene fail to develop mature osteoclasts, reflecting an absolute requirement for the c-Fos proto-oncogene in osteoclast precursors. C-Fos is also expressed in mature osteoclasts; however, the regulation of fos in these cells has not been studied. By using cultured murine osteoclast-like cells (OCLs) we found that treatment with colony-stimulation factor 1 (CSF-1) induced a 3.9-fold increase in c-Fos rnRNA at 30 minutes and a 2.6-fold increase at 60 minutes. With use of mature osteoclasts isolated from transgenic mice expressing the bacterial Lac-Z gene under the control of the murine fos promoter, we were able to directly demonstrate transcriptional activation of fos by CSF-1 in these cells. Transcriptional activation was 2.6-fold greater at 5 minutes and 2.8-fold greater at 15 minutes in CSF-1-treated cells than in vehicle-treated cells. CSF-1 induced nuclear protein binding to the fos serum response element that was significantly attenuated by antibodies to the transcription factor Elk-1 but not by Sap-1a. Treatment of mature osteoclasts with CSF-1 for 2 hours resulted in a significant increase in the levels of nuclear c-Fos protein. These data demonstrate that CSF-1 upregulates c-fos expression in mature osteoclasts at least in part via transcriptional activation of fos. CSF-1 induced binding of Elk-1 to the fos gene serum response element appears to be part of the molecular mechanism by which this occurs.

Selective deletion of the soluble Colony-Stimulating Factor 1 isoform in vivo prevents estrogen-deficiency bone loss in mice

Neutralizing CSF1 in vivo completely prevents ovariectomy (OVX)-induced bone loss in mice. There are two isoforms of CSF1, soluble (sCSF1), and membrane-bound (mCSF1), but their individual biological functions are unclear. It had been previously reported that mCSF1 knockout (K/O) and wild type (Wt) female mice experience the same degree of bone loss following OVX. In Wt mice the expression of sCSF1 was elevated fourfold in skeletal tissue following OVX while expression of mCSF1 was unchanged. To examine the role of sCSF1 in OVX-induced bone loss, mice were engineered in which sCSF1 was not expressed but expression of mCSF1 was unaffected (sCSF1 K/O). Isoform-specific reverse transcription PCR confirmed the absence of transcripts for sCSF1 in bone tissue isolated from these animals and no circulating CSF1 was detected by ELISA. Surprisingly, there were no significant differences in bone mineral density (BMD) between sCSF1 K/O mice and Wt controls as assessed by dual-energy X-ray absorptiometry and micro-CT. However, one month after OVX, femoral, spinal and total BMD had declined by 11.2%, 8.9%, and 8.7% respectively in OVX-Wt animals as compared to Sham-OVX. In contrast OVX sCSF1 K/O mice showed changes of +0.1%, −2.4%, and +2.3% at the same 3 sites compared to Sham-OVX sCSF1 K/O mice. These data indicate important non-redundant functions for the two isoforms of CSF1 and suggest that sCSF1, but not mCSF1, plays a key role in estrogen-deficiency bone loss.

An Unusual Case of Rickets and How Whole Exome Sequencing Helped to Correct a Diagnosis

ABSTRACT Objective: Rickets can be caused by a wide variety of underlying nutritional deficiencies or genetic defects. There is significant overlap in the clinical presentations of the various forms of rickets and the risk for misdiagnosis is great. This report describes a diagnostically challenging case of rickets that highlights important differences between two forms of rickets and demonstrates how gene sequencing technology can be instrumental in the identification and further characterization of these diseases. Methods: A patient diagnosed with X-linked hypophosphatemia (XLH) as a toddler did not develop many of the classic clinical features of this disease over time, calling the diagnosis into question. Whole exome sequencing (WES) was performed in this setting to determine the genetic basis of disease. Results: WES revealed that the patient did not have any mutations associated with XLH and instead was a compound heterozygote for 2 frameshift mutations in CYP27B1, leading to a revised diagnosis of ...