Regulation of SDF-1 (CXCL12) production by osteoblasts; a possible mechanism for stem cell homing.

Stromal derived factor-1 (SDF-1 or CXCL12) controls many aspects of stem cell function including trafficking and proliferation. Previously, it was demonstrated that DNA-damaging agents such as irradiation, cyclophosphamide or 5-fluorouracil increase the expression of SDF-1 by osteoblasts in murine marrow. Here, the production of SDF-1 by osteoblasts in vitro in response to cytokines known to be particularly important in bone physiology was examined using primary human osteoblasts (HOBs), mixed marrow stromal cells (BMSCs), and by, mouse, rat and human osteoblast-like cell lines. From these studies, it was determined that the expression of SDF-1 is an early feature of osteoblastic induction that may be modulated by IL-1beta, PDGF-BB, VEGF, TNF-alpha and PTH. Each of these factors increased SDF-1 synthesis, while TGF-beta1 decreased SDF-1 secretion. Of note, the biodistribution of SDF-1 in culture was equally distributed between the medium and detergent-soluble and -insoluble fractions of the cultures. Immunohistochemistry of developing bones demonstrated that SDF-1 was also a feature of early bone development first beginning in the perichondrium and moving into the marrow cavity of the developing bone analogue. As SDF-1 expression increases in response to PTH in vitro, animals were treated with an anabolic regime of PTH for 21 days. Under these conditions, significant increases in SDF-1 mRNA expression were observed near the growth plate and epiphysis regions of the long bones. Yet, in serum, immunodetectable SDF-1 levels were significantly reduced (24%) in the PTH-treated animals (Vehicle: 408 +/- 25 vs. PTH 308 +/- 20 SDF-1 pg/ml). Together, these data suggest a possible mechanism for localizing stem cells into a developing marrow where increased expression of SDF-1 in the local marrow environment along with decreased SDF-1 in the serum may create a homing gradient.

Parathyroid hormone stimulates fra-2 expression in osteoblastic cells in vitro and in vivo.

PTH and PTH-related protein (PTHrP) are key mediators of skeletal development and homeostasis through their activation of the PTH-1 receptor. Previous studies have found that several AP-1 family members are regulated by PTH, such as c-fos, fra-1, and c-jun. There are numerous genes in the bone microenvironment that contain AP-1 sites, and different Fos family members are reported to have opposing transcriptional activities at AP-1 sites. The purpose of this study was to identify the effects of PTH on expression of the AP-1 protein complex member, fra-2, to extend our understanding of transcriptional regulators of PTH action. PTH induction of fra-2 messenger RNA (mRNA) levels in MC3T3-E1 preosteoblastic cells was maximal with 0.1 microM PTH (1-34). The expression in vitro was greatest 1 h after treatment and was present with N-terminal PTH but not PTH (7-34) or (53-84). Cycloheximide treatment induced fra-2 expression, and actinomycin D inhibited basal and PTHrP-induced expression. AP-1 protein in nuclear extracts of MC3T3-E1 cells was increased with PTH treatment at 3 h and consisted of high levels of Fra-2 protein, as evidenced by a supershift in an electrophoretic mobility shift assay and Western blot analysis. Up-regulation of steady-state fra-2 mRNA was also noted in vivo, where injection of PTH (1-34) (20 microgram) resulted in a more-than-7-fold maximal increase in fra-2 mRNA expression in the calvaria of mice, after 1 h of treatment. These data add to the transcriptional mediators induced by PTH and suggest that the interplay of AP-1 family members will provide insight into regulatory pathways of PTH and PTHrP for their anabolic and catabolic actions in bone.

Effects of sex steroid receptor specificity in the regulation of skeletal metabolism.

The interaction between estrogens and androgens, with their protective effects in bone, and parathyroid hormone (PTH), a calcitropic peptide hormone, is complex but may be better understood with murine models. The purpose of this study was to characterize skeletal phenotypes of mice deficient in estrogen receptor alpha (ERalpha), androgen receptor (AR, mutant tfm), or both, and determine if ERalpha and AR alter osteoblast differentiation and/or PTH response in vitro. Loss of ERalpha resulted in increased long bone length in females, but reduced length in males, suggesting loss of ERalpha reversed sex steroid-dependent skeletal dimorphism. The AR deficient tfm mice (genetically male but phenotypically female) had the longest bones and, similar to males, lengths were reduced with loss of ERalpha. Loss of AR and/or ERalpha resulted in a reduction in femoral bone mineral density (BMD) compared to male wildtype (WT) mice, suggesting tfm mice follow the female sex for BMD. In males or tfm mice, but not females, loss of AR and/or ERalpha caused a reduction in cortical width of the tibia compared to male WT mice. Reduced trabecular bone was found in tibiae of female and tfm mice versus male littermates, suggesting that tfm mice follow the female sex for trabecular bone but loss of ERalpha did not alter trabecular bone levels. Primary calvarial osteoblasts of male WT mice were less responsive to PTH stimulation of cAMP than all other genotypes, suggesting the female chromosomal sex and/ or loss of ERalpha or AR results in increased sensitivity to PTH. In conclusion, tfm mice follow the male pattern of long bone development, but imitate females in bone density and trabecular bone. Loss of ERalpha and/or AR results in increased osteoblast sensitivity to PTH and may explain actions of PTH noted in hypogonadal humans.

Transgenic models of metabolic bone disease: impact of estrogen receptor deficiency on skeletal metabolism.

Estrogen has protective effects on the skeleton via its inhibition of bone resorption. Mechanisms for these effects and the selectivity to the estrogen receptor alpha (ER alpha) or ER beta are unclear. The purpose of our study was to determine the impact of the ER alpha on skeletal metabolism using murine models with targeted disruption of the ER alpha and beta. Mice generated by homologous recombination and Cre/loxP technology yielding a deletion of the ER alpha exon 3 were evaluated and also crossed with mice with a disruption of the exon 3 of the ER beta to result in double ER alpha and ER beta knockout mice. Skeletal analysis of long bone length and width, radiographs, dual X-ray absorptiometry, bone histomorphometry, micro computerized tomography, biomechanical analysis, serum biochemistry, and osteoblast differentiation were evaluated. Male ER alpha knockout mice had the most dramatic phenotype consisting of reduced bone mineral density (BMD), and bone mineral content (BMC) of femurs at 10 and 16 weeks and 8-9 months of age. Female ER alpha knockout mice also had reduced density of long bones but to a lesser degree than male mice. The reduction of trabecular and cortical bone in male ER alpha knockout mice was statistically significant. Male double ER alpha and ER beta knockouts had similar reductions in bone density versus the single ER alpha knockout mice suggesting that the ER alpha is more protective than the ER beta in bone. In vitro analysis revealed no differences in osteoblast differentiation or mineralized nodule formation among cells from ER alpha genotypes. These data suggest that estrogens are important in skeletal metabolism in males; the ER alpha plays an important role in estrogen protective effects; osteoblast differentiation is not altered with loss of the ER alpha; and compensatory mechanisms are present in the absence of the ER alpha and/or another receptor for estrogen exists that mediates further effects of estrogen on the skeleton.