Turner, Russell T.

The decrease in cancellous bone formation after estrogen treatment is generally thought to be coupled with a prior decrease in bone resorption. To test the possibility that estrogen has rapid tissue-specific actions on bone metabolism, we determined the time course (1–32 h) effects of diethylstilbestrol on steady-state mRNA levels for immediate-response genes, extracellular matrix proteins, and signaling peptides in the proximal tibial metaphysis and uterus by using Northern blot and RNase protection assays. The regulation of signaling peptides by estrogen, although tissue specific, followed a similar time course in bone and uterus. The observed rapid decreases in expression of insulin-like growth factor I, a growth factor associated with bone formation; decreases in mRNA levels for bone matrix proteins; evidence for reduced bone matrix synthesis; failure to detect rapid increases in mRNA levels for signaling peptides implicated in mediating the inhibitory effects of estrogen on bone resorption (interleukin-1 and -6) as well as other cytokines that can increase bone resorption; and the comparatively long duration of the bone remodeling cycle in rats indicate that estrogen can decrease bone formation by a mechanism that does not require a prior reduction in bone resorption.

Total cellular RNA was extracted from bone cells of three different femoral compartments of 2-mo-old rats. The intact femora were first incubated with collagenase to obtain periosteal cells. The bisected periosteum-free diaphyses and metaphyses were then incubated with collagenase to obtain enriched populations of endosteal and cancellous bone cells, respectively. The total cellular RNA from these three tissues was separated by size using agarose gel electrophoresis, transferred to nylon filters, hybridized to 32P-labeled cDNA probes for glyceraldehyde-3-phosphate dehydrogenase (GAP), pre-pro-alpha (I) type I collagen (collagen), osteocalcin (BGP), and alkaline phosphatase (AP), and the cDNA/mRNA hybrids were visualized by radioautography. Bone matrix deposition was measured in each tissue compartment by tetracycline-based dynamic bone histomorphometry. The bone formation and apposition rates were greatest in the periosteum and least in metaphysis. Mean mRNA levels for collagen and BGP were positively correlated with mean bone formation and mineral apposition rates. Interestingly, mean AP mRNA levels were not correlated with indexes of bone formation. These results demonstrate that the steady-state mRNA levels for bone matrix proteins in femora show pronounced site specificity and correlate with the rates of bone matrix deposition.

The effects of dehydroepiandrosterone (DHA) on cortical bone at the tibial diaphysis and cancellous bone at the tibial metaphysis were determined in intact and ovariectomized rats. Total plasma DHA was reduced at 10 and 16 wk after ovariectomy. Intact and ovariectomized rats were implanted monthly with controlled-release subcutaneous pellets containing 2.5 mg of DHA or placebo. Plasma DHA was elevated in DHA-treated intact rats and was near normal in hormone-treated ovariectomized rats. Ovariectomy resulted in severe cancellous bone osteopenia, which was reduced in DHA-treated animals. Cross-sectional and cortical areas were increased at the diaphysis in ovariectomized rats, and this was not altered by DHA treatment. Bone formation at the endosteal and periosteal surfaces of the tibial diaphysis was decreased 16 wk after ovariectomy, and DHA treatment appeared to antagonize these changes. We interpret these results as evidence that decreased peripheral DHA levels contribute to the skeletal changes in ovariectomized rats.

This report describes the relationship between bone formation and mRNA levels for selected bone proteins. Dynamic bone histomorphometry was used to measure bone formation in tibial periosteum of male rats from weanling (3 wk) to 52 wk old. Northern blot analysis of freshly isolated periosteal cells from the long bones was used to determine steady-state mRNA levels for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAP), the bone matrix proteins osteocalcin (BGP), and prepro-alpha-2 (I) chain of type 1 precollagen (collagen), the osteoblast marker enzyme alkaline phosphatase (AP), and the osteoblast-derived signaling factor (growth factor) transforming growth factor-beta (TGF-beta). Radial growth at the tibial diaphysis achieved a maximum value in 8-wk-old rats and decreased progressively with age thereafter. This age-related decrease in the radial growth rate was initially due to reduced osteoblast activity; however, in older rats (greater than 17 wk old) reduced osteoblast number contributed to the decrease in bone formation. There was a strong correlation between the steady-state mRNA level for collagen and the periosteal bone formation rate. In contrast, the mRNA levels for the other bone proteins were more weakly correlated (TGF-beta and AP) or not correlated (BGP). These results suggest that the decreased bone matrix synthesis by periosteal cells in long bones of maturing rats is due to decreased expression of genes for bone matrix proteins.

Osteolytic bone diseases, such as osteoporosis, are characterized by diminished bone quality and increased fracture risk. The therapeutic challenge remains to maintain bone homeostasis with a balance between osteoclast-mediated resorption and osteoblast-mediated formation. Osteoclasts are formed by the fusion of monocyte/macrophage-derived precursors. Here we report, to our knowledge for the first time, that receptor-interacting protein 140 (RIP140) expression in osteoclast precursors and its protein regulation are crucial for osteoclast differentiation, activity, and coupled bone formation. In mice, monocyte/macrophage–specific knockdown of RIP140 (mϕRIP140KD) resulted in a cancellous osteopenic phenotype with significantly increased bone resorption and reduced bone formation. Osteoclast precursors isolated from mϕRIP140KD mice had significantly increased differentiation potential. Furthermore, conditioned media from mϕRIP140KD primary osteoclast cultures significantly suppressed osteoblast differentiation. This suppressive activity was effectively and rapidly terminated by specific Syk-stimulated RIP140 protein degradation. Mechanistic analysis revealed that RIP140 functions primarily by inhibiting osteoclast differentiation through forming a transcription-suppressor complex with testicular receptor 4 (TR4) to repress osteoclastogenic genes. These data reveal that monocyte/macrophage RIP140/TR4 complexes may serve as a critical transcription regulatory complex maintaining homeostasis of osteoclast differentiation, activity, and coupling with osteoblast formation. Accordingly, we propose a potentially novel therapeutic strategy, specifically targeting osteoclast precursor RIP140 protein in osteolytic bone diseases.

Abstract A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-α2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-β, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.