The skeleton is an efficient 'servo' (feedback-controlled/steady-state) system that continuously integrates signals and responses which sustain its functions of delivering calcium while maintaining strength. In many individuals, bone mass homeostasis starts failing in midlife, leading to bone loss, osteoporosis and debilitating fractures. Recent advances, spearheaded by genetic information, offer the opportunity to stop or reverse this downhill course.

PGE, and PGE2 are potent stimulators of bone formation. Osteogenesis is strongly dependent on angiogenesis. Vascular endothelial growth factor (VEGF), a secreted endothelial cellspecific mitogen, has been implicated in physiological and pathological angiogenesis. The aim of this study was to examine the possible role of VEGF in PG stimulation of bone formation. We found that in rat calvaria-derived osteoblast-enriched cells and in the osteoblastic RCT-3 cell line PGE2 and El increased VEGF mRNA and protein levels. The increased expression of VEGF mRNA produced by PGE2 was rapid (maximal at 1 h), transient (declined by 3 h), potentiated by cycloheximide, and abolished by actinomycin D. PGE2 had no effect on VEGF mRNA stability, suggesting transcriptional regulation ofVEGF expression by PGE2. Rp-cAMP, a cAMP antagonist, suppressed VEGF mRNA induced by PGE2, indicating cAMP mediation. The upregulation of VEGF expression by PGE2 in the preosteoblastic RCT-1 cells was potentiated by treatment with retinoic acid, which induces the differentiation of these cells. The upregulation of VEGF mRNA by PGE2 was inhibited by dexamethasone treatment. In addition, Northern blot analysis showed that VEGF mRNA is expressed in adult rat tibia. In summary, we documented, for the first time, the expression of VEGF in osteoblasts and in bone tissue. Stimulation of VEGF expression by PGs and its suppression by glucocorticoids, which, respectively, stimulate and suppress bone formation, strongly implicate the involvement of VEGF in bone metabolism. (J. Clin. Invest. 1994.93:2490-2496

Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.

The androgen receptor (AR), when complexed with 5␣-dihydrotestosterone (DHT), supports the survival and proliferation of prostate cells, a process critical for normal development, benign prostatic hypertrophy, and tumorigenesis. However, the androgen-responsive genetic pathways that control prostate cell division and differentiation are largely unknown. To identify such pathways, we examined gene expression in the ventral prostate 6 and 24 h after DHT administration to androgen-depleted rats. 234 transcripts were expressed significantly differently from controls (p < 0.05) at both time points and were subjected to extensive data mining. Functional clustering of the data reveals that the majority of these genes can be classified as participating in induction of secretory activity, metabolic activation, and intracellular signaling/signal transduction, indicating that AR rapidly modulates the expression of genes involved in proliferation and differentiation in the prostate. Notably AR represses the expression of several key cell cycle inhibitors, while modulating members of the wnt and notch signaling pathways, multiple growth factors, and peptide hormone signaling systems, and genes involved in MAP kinase and calcium signaling. Analysis of these data also suggested that p53 activity is negatively regulated by AR activation even though p53 RNA was unchanged. Experiments in LNCaP prostate cancer cells reveal that AR inhibits p53 protein accumulation in the nucleus, providing a post-transcriptional mechanism by which androgens control prostate cell growth and survival. In summary these data provide a comprehensive view of the earliest events in AR-mediated prostate cell proliferation in vivo, and suggest that nuclear exclusion of p53 is a critical step in prostate growth.

Dehydroepiandrosterone (DHEA) exhibits peak adrenal secretion in the fetus at term and around age 30 yr in the adult. Levels then progressively decline, which is associated with decreased levels of testosterone, dihydrotestosterone, and estrogen in peripheral tissues. DHEA supplementation in postmenopausal women increases bone formation and density, an effect mainly attributed to peripheral conversion to sex hormones. In this study, we tested DHEA for direct effects on the androgen (AR) and estrogen (ER) receptors. DHEA bound to AR with a Ki of 1 microM, which was associated with AR transcriptional antagonism on both the mouse mammary tumor virus and prostate-specific antigen promoters, much like the effects of bicalutamide. Unlike bicalutamide, DHEA stimulated, rather than inhibited, LNCaP cell growth, suggesting possible interaction with other hormone receptors. Indeed DHEA bound to ERalpha and ERbeta, with Ki values of 1.1 and 0.5 microM, respectively. Despite the similar binding affinities, DHEA showed preferential agonism of ERbeta with an EC50 of approximately 200 nm and maximal activation at 1 microM. With ERalpha we found 30-70% agonism at 5 microM, depending on the assay. Physiological levels of DHEA are approximately 30 nM and up to 90 nM in the prostate. DHEA at 30 nM is actually sufficient to activate ERbeta transcription to the same degree as estrogen at its circulating concentration, and additive effects are seen when the two were combined. Taken together, DHEA has the potential for physiologically relevant direct activation of ERbeta. With peak levels at term and age 30 yr, there is also a potential for antagonist effects on AR and partial agonism of ERalpha.

A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor 131, or platelet-derived growth factor

Sox8 encodes a high-mobility group transcription factor that is widely expressed during development. Sox8, -9 and -10 form group E of the Sox gene family which has been implicated in several human developmental disorders. In contrast to other SoxE genes, the role of Sox8 is unclear and Sox8 mouse mutants reportedly showed only idiopathic weight loss and reduced bone density. The careful analysis of our Sox8 null mice, however, revealed a progressive male infertility phenotype. Sox8 null males only sporadically produced litters of reduced size at young ages. We have shown that SOX8 protein is a product of adult Sertoli cells and its elimination results in an age-dependent deregulation of spermatogenesis, characterized by sloughing of spermatocytes and round spermatids, spermiation failure and a progressive disorganization of the spermatogenic cycle, which resulted in the inappropriate placement and juxtaposition of germ cell types within the epithelium. Those sperm that did enter the epididymides displayed abnormal motility. These data show that SOX8 is a critical regulator of adult Sertoli cell function and is required for both its cytoarchitectural and paracrine interactions with germ cells.

Homology searches for HLH factors that play a similar role in adipocyte, osteoblast, or chon-Merck Research Laboratories West Point, Pennsylvania 19486 droblast differentiation have not been successful (negative data are rarely published). However, a key regulatory transcription factor in adipocyte differentiation, belonging to the nuclear receptor family (peroxisome prolifera-The mineralized skeleton was essential for the evolution tor activated receptor ␥2 or PPAR␥2; Tontonoz et al., of terrestrial vertebrates since it provided an ample 1994), was discovered three years ago. A transcription source of calcium and phosphate and facilitated locofactor that plays a key role in osteoblast differentiation motion on solid ground. Bone also provides protection is described in this issue of Cell. This factor is CBFA1, from external injury for the brain and spinal cord. In a member of the core-binding factor family, also known addition, it houses and interacts with the bone marrow, as PEBP2␣A (for polyoma enhancer binding protein) and supporting hematopoieses. Another crucial mechanical as AML3 (for acute myeloid leukemia). function of bone in land vertebrates is breathing, and CBFA belongs to the Runt domain gene family, homol-a severely compromised skeleton causes immediate ogous to the Drosophila melanogoster pair-rule gene postnatal death.runt that plays a role in the formation of the segmented Embryologically, we recognize two types of bone. Inbody pattern as well as in sex determination and in tramembraneous bone is formed directly by bone cells the development of the nervous system (Ogawa et al., within connective tissue. It originates from the branchial 1993). The ␣ subunit of these heterodimeric transcription arches and gives rise to the bones of the skull and the factors binds to DNA via the Runt domain when paired face. The rest of the skeleton is generated by bone with the ␤ subunit, called CBFB/PEBP2␤, which does replacement of a cartilage anlage through the process not directly interact with DNA. The consensus DNA bind-of endochondral ossification, where vascular invasion ing sequence is PuACCPuCA, originally identified as an is followed by the deposition of bone on mineralized enhancer in polyoma virus and murine leukemia virus, cartilage, which is subsequently removed. This bone is but also present in many T cell-specific genes, TCR␣, derived from the somites that contain multipotent cells -␤, -␦, -␥, CD3⑀, as well as in genes encoding the en-capable of differentiating into muscle, dermis, and cartizymes myeloperoxidase, neutrophil elastase, and gran-lage. Experimental approaches have identified pluripozyme B, and in several genes encoding cytokines and tent mesenchymal cells that have myogenic, chondrotheir receptors GM-CSF, IL-3, and CSF-1. genic, and adipogenic potential, raising the question of Three CBFA genes have been identified: CBFA1/ signals and regulatory pathways that determine commit-PEBP2␣A/AML3, CBFA2/PEB2␣B/AML1, and CBFA3/ ment to one of these lineages. In vivo, the cues seem PEBP2␣C/AML2...