Effects of fibroblast growth factor-2 on longitudinal bone growth.

In vivo, fibroblast growth factor-2 (FGF-2) inhibits longitudinal bone growth. Similarly, activating FGF receptor 3 mutations impair growth in achondroplasia and thanatophoric dysplasia. To investigate the underlying mechanisms, we chose a fetal rat metatarsal organ culture system that would maintain growth plate histological architecture. Addition of FGF-2 to the serum-free medium inhibited longitudinal growth. We next assessed each major component of longitudinal growth: proliferation, cellular hypertrophy, and cartilage matrix synthesis. Surprisingly, FGF-2 stimulated proliferation, as assessed by [3H]thymidine incorporation. However, autoradiographic studies demonstrated that this increased proliferation occurred only in the perichondrium, whereas decreased labeling was seen in the proliferative and epiphyseal chondrocytes. FGF-2 also caused a marked decrease in the number of hypertrophic chondrocytes. To assess cartilage matrix synthesis, we measured 35SO4 incorporation into newly synthesized glycosaminoglycans. Low concentrations (10 ng/ml) of FGF-2 stimulated cartilage matrix production, but high concentrations (1000 ng/ml) inhibited matrix production. We conclude that FGF-2 inhibits longitudinal bone growth by three mechanisms: decreased growth plate chondrocyte proliferation, decreased cellular hypertrophy, and, at high concentrations, decreased cartilage matrix production. These effects may explain the impaired growth seen in patients with achondroplasia and related skeletal dysplasias.

Visualization of fibrous dysplasia during somatostatin receptor scintigraphy.

Somatostatin receptor scintigraphy was performed on a patient with McCune-Albright syndrome and acromegaly. No evidence of pituitary disease was found, but uptake of (111)In-pentetreotide was noted in areas of fibrous dysplasia. This uptake was not changed after 6 mo of octreotide therapy. The patient's bone disease also remained stable. The possible implications of these findings are discussed.

Absence of androgen-mediated transcriptional effects in osteoblastic cells despite presence of androgen receptors.

Androgen excess and deficiency affect skeletal maturation and bone cell function. Understanding the molecular basis for these androgen effects could improve therapy/prevention of short stature and osteoporosis. Androgens act through binding to androgen receptors (ARs), which modulate gene transcription via interactions with DNA response elements on target genes. Because osteoblasts contain ARs at levels just below certain androgen-sensitive tissues, we sought to define the function of AR in a number of commonly used osteoblastic cell lines. Presence and quantification of AR protein and mRNA were evaluated by ligand binding assay, western blotting, and RNAse protection assay. AR-containing osteoblastic cell lines were exposed to nonaromatizable androgens and effects on gene expression were assessed. We found no evidence for direct effects of androgen on endogenous genes nor was androgen involved in modulation of parathyroid hormone effects on early gene activation. Androgen-sensitive reporter gene constructs were stimulated by androgen only when AR cDNA expression vectors were introduced into cells by cotransfection. We conclude that, in commonly used osteoblastic cell lines, the presence of AR at the levels described here does not guarantee androgen transcriptional activity. The effects of androgen on bone in vivo may involve direct stimulation of osteoblastic cells in a different setting or stage of differentiation. Alternatively, androgen may act on bone cells other than osteoblasts, or through metabolic conversion to estrogens.

Factitious Cushing syndrome.

There have been few reports of factitious Cushing syndrome. To characterize the clinical and laboratory features leading to this unusual diagnosis, we describe 6 patients (5 women, 1 man), ages 31-44, identified retrospectively among 860 patients evaluated for hypercortisolism at the National Institutes of Health Clinical Center. All six patients had multiple surgeries unrelated to Cushing syndrome and a history of depression or anxiety. Four patients had close contact with the medical profession, three a history of drug abuse, and three had undergone previous treatment for Cushing syndrome. The physical features of Cushing syndrome were variable and not helpful in the differential diagnosis with endogenous Cushing syndrome. Four patients had striking variability in urine-free cortisol (UFC) and 17-hydroxysteroid (17-OHCS) values from low to high. Adrenal computed tomography, performed in two patients, showed small adrenal glands (n = 1) or a left-sided mass (n = 1), and adrenal magnetic resonance imaging, performed in one patient, showed atrophic glands. Pituitary magnetic resonance imaging, carried out in four patients, was either normal (n = 1) or exhibited questionable signs of microadenoma (n = 3). Determination of synthetic glucocorticoids by high pressure liquid chromatography (HPLC) was positive in the four patients in whom it was performed. Factitious Cushing syndrome is a difficult diagnosis. To conserve time and resources, high pressure liquid chromatography analysis of urine steroids, the most definitive test for the factitious disorder, should be performed whenever there is clinical suspicion of glucocorticoid abuse.

Effects of hormones and intracellular mediators on differentiated functions of cultured Leydig tumor cells.

Cellular regulation by hormones that utilize a myriad of intracellular signaling pathways is recognized to be quite complex. To investigate some of these effects in an established cell line, we tested a panel of hormones and modulators for their effects on cyclic AMP (cAMP) and progesterone production, both alone and in combination with human chorionic gonadotropin (hCG), using the MA-10 cultured Leydig tumor cell line. None significantly affected intracellular levels of cAMP, and only epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated progesterone production. While EGF, basic fibroblast growth factor, insulin, insulin-like growth factor-1, and transforming growth factor beta all decreased cAMP production only, TPA decreased hCG-stimulated cAMP and progesterone production. Those factors that stimulated progesterone production also induced a characteristic morphological change ("rounding") of these cells. In addition, EGF, insulin, and TPA, like hCG, elevated mRNA levels of competence oncogenes (c-fos and c-myc), albeit to different extents. These data demonstrate the wide range of hormones to which the cultured Leydig tumor cell will respond, as well as the varying degree of responses observed in the intracellular signaling pathways that we examined.

Transiently elevated levels of c-fos and c-myc oncogene messenger ribonucleic acids in cultured murine Leydig tumor cells after addition of human chorionic gonadotropin.

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.

The regulation of granulosa cell proopiomelanocortin messenger ribonucleic acid by androgens and gonadotropins.

We have previously demonstrated the presence of proopiomelanocortin (POMC) messenger RNA (mRNA) in rat granulosa cells. This study examines the unique, tissue-specific regulation of granulosa cell POMC mRNA levels by hormones with established regulatory effects on these cells. Northern blot analysis of immature rat ovarian RNA revealed the presence of a single mRNA of approximately 900 base pairs in length. The levels of ovarian POMC mRNA increased approximately 6-fold 48 h after priming with PMSG when expressed per microgram of total RNA. Granulosa cells were isolated from immature PMSG-primed rats and cultured under serum-free conditions in the presence and absence of various hormones to determine their effect on POMC mRNA levels. Treatment of the cells for 48 h with LH (100 ng/ml), androstenedione (10(-7) M), or LH plus androstenedione elevated POMC mRNA levels. LH alone elicited a 5-fold increase in POMC mRNA, and androstenedione elicited a 10-fold increase; the combination treatment led to a 47-fold increase. The effects of the nonaromatizable androgen dihydrotestosterone (DHT) were also evaluated. Treatment for 48 h with DHT (10(-7) M) elicited a 40-fold increase in POMC mRNA levels. In vivo experiments measuring ovarian POMC mRNA from immature female rats corroborated the in vitro results. Animals injected sc with DHT (500 micrograms) demonstrated 5-fold increases in ovarian POMC mRNA when expressed per microgram of total RNA. These studies provide both in vitro and in vivo evidence that granulosa cell POMC mRNA is under unique hormonal regulation by both androgens and gonadotropins.