FGF2 crosstalk with Wnt signaling in mediating the anabolic action of PTH on bone formation.

The mechanisms of the anabolic effect of parathyroid hormone (PTH) in bone are not fully defined. The bone anabolic effects of PTH require fibroblast growth factor 2 (FGF2) as well as Wnt signaling and FGF2 modulates Wnt signaling in osteoblasts. In vivo PTH administration differentially modulated Wnt signaling in bones of wild type (WT) and in mice that Fgf2 was knocked out (Fgf2KO). PTH increased Wnt10b mRNA and protein in WT but not in KO mice. Wnt antagonist SOST mRNA and protein was significantly higher in KO group. However, PTH decreased Sost mRNA significantly in WT as well as in Fgf2KO mice, but to a lesser extent in Fgf2KO. Dickhopf 2 (DKK2) is critical for osteoblast mineralization. PTH increased Dkk2 mRNA in WT mice but the response was impaired in Fgf2KO mice. PTH significantly increased Lrp5 mRNA and phosphorylation of Lrp6 in WT but the increase was markedly attenuated in Fgf2KO mice. PTH increased ß-catenin expression and Wnt/ß-catenin transcriptional activity significantly in WT but not in Fgf2KO mice. These data suggest that the impaired bone anabolic response to PTH in Fgf2KO mice is partially mediated by attenuated Wnt signaling.

Sirtuin 1 is a negative regulator of parathyroid hormone stimulation of matrix metalloproteinase 13 expression in osteoblastic cells: role of sirtuin 1 in the action of PTH on osteoblasts.

Parathyroid hormone (PTH) is the only current anabolic treatment for osteoporosis in the United States. PTH stimulates expression of matrix metalloproteinase 13 (MMP13) in bone. Sirtuin 1 (SIRT1), an NAD-dependent deacetylase, participates in a variety of human diseases. Here we identify a role for SIRT1 in the action of PTH in osteoblasts. We observed increased Mmp13 mRNA expression and protein levels in bone from Sirt1 knock-out mice compared with wild type mice. PTH-induced Mmp13 expression was significantly blocked by the SIRT1 activator, resveratrol, in osteoblastic UMR 106-01 cells. In contrast, the SIRT1 inhibitor, EX527, significantly enhanced PTH-induced Mmp13 expression. Two h of PTH treatment augmented SIRT1 association with c-Jun, a component of the transcription factor complex, activator protein 1 (AP-1), and promoted SIRT1 association with the AP-1 site of the Mmp13 promoter. This binding was further increased by resveratrol, implicating SIRT1 as a feedback inhibitor regulating Mmp13 transcription. The AP-1 site of the Mmp13 promoter is required for PTH stimulation of Mmp13 transcriptional activity. When the AP-1 site was mutated, EX527 was unable to increase PTH-stimulated Mmp13 promoter activity, indicating a role for the AP-1 site in SIRT1 inhibition. We further showed that SIRT1 deacetylates c-Jun and that the cAMP pathway participates in this deacetylation process. These data indicate that SIRT1 is a negative regulator of MMP13 expression, SIRT1 activation inhibits PTH stimulation of Mmp13 expression, and this regulation is mediated by SIRT1 association with c-Jun at the AP-1 site of the Mmp13 promoter.

Fibroblast growth factor-2, bone homeostasis and fracture repair.

There remains a great need to develop therapeutic agents to treat critical size defects and non-union fractures and one of the potential agents is recombinant human fibroblast growth factor 2 (FGF-2). We discuss the function of FGF-2 in bone formation, bone resorption, and downstream signaling pathways and review the role of exogenous FGF-2 in fracture healing. The importance of endogenous FGF-2 in bone formation and its potential importance in fracture healing in response to parathyroid hormone (PTH) and bone morphogenetic protein 2 (BMP2) is described. In addition we will review, FGF-2 signaling crosstalk with Wnt signaling and PTH signaling in bone formation and repair. Finally, we discuss the outstanding unresolved issues in the application of FGF-2 as therapeutic agent for bone regeneration.

Role of fibroblast growth factor 2 and Wnt signaling in anabolic effects of parathyroid hormone on bone formation.

Osteoporosis poses enormous health and economic burden worldwide. One of the very few anabolic agents for osteoporosis is parathyroid hormone (PTH). Although great progress has been made since the FDA approved PTH in 2002, the detailed mechanisms of the bone anabolic effects of intermittent PTH treatment is still not well understood. PTH bone anabolic effect is regulated by extracellular factors. Maximal bone anabolic effect of PTH requires fibroblast growth factor 2 (FGF2) signaling, which might be mediated by transcription factor activating transcription factor 4 (ATF4). Maximal bone anabolic effect of PTH also requires Wnt signaling. Particularly, Wnt antagonists such as sclerostin, dickkopf 1 (DKK1) and secreted frizzled related protein 1 (sFRP1) are promising targets to increase bone formation. Interestingly, FGF2 signaling modulates Wnt/ß-Catenin signaling pathway in bone. Therefore, multiple signaling pathways utilized by PTH are cross talking and working together to promote bone formation. Extensive studies on the mechanisms of action of PTH will help to identify new pathways that regulate bone formation, to improve available agents to stimulate bone formation, and to identify potential new anabolic agents for osteoporosis.

Stem cell-based bone repair.

To accelerate bone repair, one strategy is to deliver the cells that make bone. The current review focuses on stem cell-based bone repair. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can self-renew unlimitedly and differentiate into the bone forming cells - osteoblasts. Scientists have been actively investigating culture conditions to stably and efficiently induce differentiation of these stem cells into osteoblasts. However, ESCs have the issues of ethnics, immune response and both ESCs and iPSCs have tumorigenic potential. In contrast, bone marrow stromal/stem cells (BMSCs) hold great potential to enhance bone formation. Use of BMSCs can avoid the ethical issues and can obviate the immune response problem. However, BMSCs are a rare population with limited self-renewal ability and their differentiation ability decreases in elderly individuals. Considering the unlimited self-renewal ability, it is promising to develop protocols to differentiate ESCs into osteoblasts faithfully and efficiently. It is important to eliminate undifferentiated ESCs or iPSCs because of their tumorigenic potential. Therefore, future studies need to identify BMSCs specific cell surface markers since the cell surface markers utilized currently are not specific to BMSCs. Future studies also need to enhance the osteogenic potential without using viral vectors for transgene delivery to eliminate the risk of tumor generation.

Fibroblast growth factor 2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway.

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/ß-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and ß-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and ß-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3ß, a negative regulator of Wnt/ß-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted ß-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.

The impaired bone anabolic effect of PTH in the absence of endogenous FGF2 is partially due to reduced ATF4 expression.

Parathyroid hormone (PTH) is currently the only approved anabolic agent for osteoporosis pharmacotherapy in the USA. However, the molecular and cellular mechanisms underlying which intermittent PTH stimulates bone formation are not fully established. Activating transcription factor 4 (ATF4) was recently identified to be a downstream target of PTH signaling in osteoblasts and FGF2 is able to rapidly increase ATF4 mRNA and protein expression in osteoblasts. Furthermore, ATF4 expression is markedly reduced in Fgf2(-/-) osteoblasts. In addition, FGF2 is required for the anabolic action of PTH on bone formation. Therefore, we hypothesize that the impaired anabolic effect of PTH in Fgf2(-/-) mice is partially due to reduced ATF4 expression. To test this hypothesis, we examined the ability of PTH to increase ATF4 expression in vitro and in vivo. In vitro data showed that PTH induced a significant increase in ATF4 mRNA expression as early as 15 min in Fgf2(+/+) primary bone marrow stromal cells (BMSCs) but not in Fgf2(-/-) BMSCs. In vivo data showed that treatment with PTH (1-34) (40 µg/kg/d) treatment for 2 weeks in 21-23 months female mice increased lumbar vertebrae bone mineral density in Fgf2(+/+) (13.8% increase). In contrast there was a 2.1% decrease in Fgf2(-/-) mice. Interestingly, basal ATF4 mRNA expression in tibiae was significantly lower in Fgf2(-/-) mice (46% decrease) compared to Fgf2(+/+) mice. PTH treatment increased ATF4 mRNA by 97% (p<0.05) in Fgf2(+/+) compared to 8% (p=0.57) in Fgf2(-/-) mice. Immunohistochemistry of vertebrae showed less ATF4 staining in Fgf2(-/-) tissue, and treatment with PTH increased ATF4 staining in Fgf2(+/+) but the increase was attenuated in Fgf2(-/-) tissue. In summary, reduced ATF4 expression may result in decreased osteoblast differentiation, and possibly contribute to the impaired stimulation of PTH on bone formation in Fgf2(-/-) mice.

Anti-resorptive effect of pilose antler blood (Cervus nippon Temminck) in ovariectomized rats.

Anti-bone resorption activity of pilose antler blood (Cervus nippon Temminck) were evaluated in ovariectomized Wistar rats. The rats were randomly divided into sham operated group (SHAM), ovariectomized group (OVX) and pilose antler blood treated group. The ovariectomized rats were treated with pilose antler blood orally in 4000 microl/kg daily doses for 10 weeks. Compared with SHAM group, serum 17 beta-estradiol level decreased significantly and osteocalcin level increased significantly in OVX group, indicating successful model of osteoporosis. The experiments showed that the bone mineral density of the lumbar spine and left femur in OVX group decreased remarkably compared to SHAM group but normalized by treatment with pilose antler blood. Additionally, serum levels of insulin-like growth factor-land testosterone were lower obviously in OVX group than those in SHAM group but preserved by pilose antler blood treatment. However, no obvious changes in serum levels of calcium, phosphorus, total alkaline phosphatase and osteoprotegerin were observed among three groups. These results suggested that administration of pilose antler blood was effective in alleviating osteoporosis in ovariectomized rats.

Fibroblast growth factor 2 positively regulates expression of activating transcription factor 4 in osteoblasts.

Our previous studies showed that basic fibroblast growth factor 2 (FGF2) null mice display markedly reduced bone mass and bone formation. However, the mechanism by which FGF2 regulates bone mass or bone formation is not fully defined. Activating transcription factor 4 (ATF4), one member of activating transcription factor/cAMP response element binding family, is a transcription factor required for osteoblast terminal differentiation. Here we investigate the ability of FGF2 to increase expression of ATF4 in bone marrow stromal cells (BMSCs) and examine ATF4 expression in Fgf2(-/-) BMSCs. We found that FGF2 stimulated ATF4 mRNA expression as early as 20 min and increased ATF4 protein expression after three hours of treatment. BMSCs from Fgf2(+/+) and Fgf2(-/-) mice were cultured in osteogenesis medium. We observed reduced alkaline phosphatase staining, decreased mineralized nodules and reduced osteocalcin expression, and reduced expression of ATF4 in Fgf2(-/-) BMSC cultures compared to Fgf2(+/+) BMSCs. This study is the first demonstration that ATF4 expression can be stimulated by FGF2 in osteoblasts and that ATF4 expression is significantly reduced in differentiated Fgf2(-/-) BMSCs. These results suggest that impaired bone mass and bone formation in Fgf2 null mice may be due in part to reduced ATF4 expression.

Reversal of osteoporotic changes of mineral composition in femurs of diabetic rats by insulin.

Insulin plays an important role in bone prevention of diabetic osteoporosis, but little is known about the relation between the bone mineral density (BMD) increase and the change of mineral element content after treated with insulin. To address this problem, male Wistar rats were randomly divided into three groups: normal group (n = 6), streptozotocin-induced diabetic group (n = 5), and streptozotocin-induced diabetic group with insulin treatment (n = 5). The femoral BMD was measured by dual energy X-ray absorptiometry, and the element content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The results showed that the femoral BMD in diabetic group was significantly lower than that in normal group (P < 0.01) but restored by insulin treatment (P < 0.01 vs diabetic group). ICP-AES analysis revealed that the element content of calcium (Ca), phosphorous (P), magnesium (Mg), strontium (Sr), and potassium (K) in diabetic group were remarkably lower than those in normal group (P < 0.01) but only Ca, P, and Mg content were significantly increased compared with diabetic group (P < 0.05) after insulin treatment. However, no significant differences were observed in element zinc (Zn) content among three groups. Our findings suggested that the loss of Ca, P, Mg, Sr, and K content accounted for the lower BMD in streptozotocin-induced diabetes rats, insulin treatment could restore BMD by increasing the content of Ca, P, and Mg.

Element analysis in femur of diabetic osteoporosis model by SRXRF microprobe.

Diabetes mellitus affects bone metabolism and leads to osteopenia and osteoporosis, but its pathogenic mechanism remains unknown. To address this problem, mineral element of bone was analyzed in experimental diabetic osteoporosis model. Male Wistar rats were randomly divided into streptozotocin (STZ)-induced diabetic group (n=5) and control group (n=5). The experiment lasted 68 days and at the end of the experiment, femoral bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry and element content in femur of animals was determined by synchrotron radiation X-ray fluorescence (SRXRF) microprobe analysis technique. Results showed that femoral BMD in diabetic group was significantly lower than that in control (P<0.01). Relative mineral content of calcium (Ca), phosphorus (P) and zinc (Zn) in diabetic femurs decreased significantly compared to controls. And strontium (Sr) in diabetics reduced 11% (P=0.09). Relative content of sulfur (S) in average was statistically higher (P<0.01) in diabetics than that in controls. But no obvious difference was observed in relative content of chromium (Cr), iron (Fe), copper (Cu), and lead (Pb) between the two groups. Statistical analysis revealed that Ca correlated positively with P (R=0.85 and P<0.001), with Sr (R=0.38 and P<0.05) and with Zn (R=0.37 and P<0.05). Whereas, Zn correlated negatively with S (R=-0.40 and P<0.05). Our results reveal that loss of minerals accounts for the BMD reduction in diabetics.