Pebbles and sand on asteroid (162173) Ryugu: In situ observation and particles returned to Earth.

The Hayabusa2 spacecraft investigated the C-type (carbonaceous) asteroid (162173) Ryugu. The mission performed two landing operations to collect samples of surface and subsurface material, the latter exposed by an artificial impact. We present images of the second touchdown site, finding that ejecta from the impact crater was present at the sample location. Surface pebbles at both landing sites show morphological variations ranging from rugged to smooth, similar to Ryugu's boulders, and shapes from quasi-spherical to flattened. The samples were returned to Earth on 6 December 2020. We describe the morphology of >5 grams of returned pebbles and sand. Their diverse color, shape, and structure are consistent with the observed materials of Ryugu; we conclude that they are a representative sample of the asteroid.

The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes.

The near-Earth carbonaceous asteroid 162173 Ryugu is thought to have been produced from a parent body that contained water ice and organic molecules. The Hayabusa2 spacecraft has obtained global multicolor images of Ryugu. Geomorphological features present include a circum-equatorial ridge, east-west dichotomy, high boulder abundances across the entire surface, and impact craters. Age estimates from the craters indicate a resurfacing age of [Formula: see text] years for the top 1-meter layer. Ryugu is among the darkest known bodies in the Solar System. The high abundance and spectral properties of boulders are consistent with moderately dehydrated materials, analogous to thermally metamorphosed meteorites found on Earth. The general uniformity in color across Ryugu's surface supports partial dehydration due to internal heating of the asteroid's parent body.

Thyroid hormone modulation of the hypothalamic growth hormone (GH)-releasing factor-pituitary GH axis in the rat.

Both thyroid hormone and hypothalamic growth hormone (GH)-releasing factor (GRF) facilitate pituitary somatotroph function. However, the pathophysiological role of thyroid hormone in GRF secretion is less well understood. Thyrotoxicosis, induced by administration of thyroxine (T4) in rats, inhibited both pituitary GH levels and immunoreactive GRF secretion from incubated hypothalamus. At the highest dose of T4 given for 12 d, GRF secretion and pituitary GH decreased by 50 and 39%, respectively. Hypothyroidism induced by thyroidectomy (Tx) enhanced GRF secretion approximately twofold while depleting pituitary GH by greater than 99%. Both of these hypothalamic and pituitary effects were reversed by replacement of T4 but not human GH for 7 or 14 d. Human GH was as potent as T4 in restoring decreased body weight gains or serum insulin-like growth factor-1 levels in Tx rats. These results indicate that at both physiological and pathological concentrations in serum, thyroid hormone acts as an inhibitory modulator of GRF secretion, probably not involving a feedback mechanism through GH. A biphasic effect of thyroid hormone on pituitary GH levels appears to derive from the difference in primary target tissues of hyper- and hypothyroidism, the hypothalamus and the pituitary, respectively.

Effects of corticotropin-releasing hormone and dexamethasone on proopiomelanocortin messenger RNA level in human corticotroph adenoma cells in vitro.

The effects of corticotropin-releasing hormone (CRH) and dexamethasone on proopiomelanocortin (POMC) mRNA levels in cultured pituitary adenoma cells were studied in 10 patients with Cushing's disease. As a control, POMC mRNA levels in cells from nonadenomatous tissues were examined in four patients. Human POMC mRNA in the cells was analyzed by Northern blot hybridization. Human POMC DNA probe hybridized with only a single size class of RNA (approximately 1,200 nucleotides) from the adenoma and nonadenoma cells of each patient. The size of POMC mRNA did not change through the culture or after incubation with CRH or dexamethasone. CRH increased POMC mRNA levels in these cells in a dose- and time-dependent manner. The minimum concentration of CRH required to elevate POMC mRNA levels in these cells exposed for 15 h was 0.1 nM. The minimum duration of 1 nM CRH treatment required to increase these levels was 3 h under our conditions. Inhibitory effects of 1 and 10 micrograms/dl dexamethasone on ACTH release and POMC mRNA levels in nonadenoma cells were greater than those in adenoma cells. These results suggest the following: (a) that the mRNA in cultured pituitary adenoma cells is qualitatively the same as that in vivo; (b) that responses of mRNA levels to CRH are time- and dose-dependent; and (c) that adenoma cells resist the inhibitory effect of dexamethasone on POMC mRNA levels and ACTH release.

Corticotropin-releasing hormone, proopiomelanocortin, and glucocorticoid receptor gene expression in adrenocorticotropin-producing tumors in vitro.

To differentiate between ectopic ACTH syndrome and Cushing's disease, gene expression of corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and glucocorticoid receptor was examined in 10 pituitary adenomas (Cushing's disease) and in 10 ectopic ACTH-producing tumors. CRH increased plasma ACTH levels in all patients with Cushing's disease and in five patients with ectopic ACTH syndrome whose tumors contained CRH and CRH mRNA. In five CRH nonresponders, CRH was not detected in tumors that contained no CRH mRNA or that contained only long-size CRH mRNA. Dexamethasone (Dex) decreased plasma ACTH levels in all patients with Cushing's disease and in three patients with ectopic ACTH-producing bronchial carcinoid. These tumors contained glucocorticoid receptor mRNA. CRH increased and Dex decreased ACTH release and POMC mRNA levels in pituitary adenoma and bronchial carcinoid cells. PMA increased POMC mRNA levels only in carcinoid cells. These results reveal characteristics of ectopic ACTH-producing tumors: long-size CRH mRNA and PMA-induced POMC gene expression. In addition, there are two ectopic ACTH syndrome subtypes: tumors containing ACTH with CRH (CRH responder) and tumors without CRH. Dex decreases ACTH release and POMC mRNA levels in some bronchial carcinoids. Therefore, CRH and Dex tests have limited usefulness in differentiating between Cushing's disease and ectopic ACTH syndrome.

Stress-induced activation of neuronal activity and corticotropin-releasing factor gene expression in the paraventricular nucleus is modulated by glucocorticoids in rats.

Intronic in situ hybridization methodology provides a means of determining the rate of gene transcription under basal and stimulated conditions. In the present study, we have used intronic in situ hybridization to the corticotropin-releasing factor (CRF) gene to measure hypothalamic CRF gene transcription after stress as well as its modulation by glucocorticoids. Using this and conventional exonic in situ hybridization we examined the time course of changes in c-fos mRNA, and CRF heteronuclear RNA (hnRNA) and mRNA concentrations in the paraventricular nucleus (PVN) of male Wistar rats after restraint stress. In addition, we determined the effects of adrenalectomy and dexamethasone administration on c-fos and CRF gene expression in the PVN. Restraint stress induced a rapid induction (within 5 min) of c-fos mRNA and CRF hnRNA expression in the PVN. Both RNA concentrations peaked at 30 min then decreased and were undetectable 2 h after stress onset. In contrast, the concentration of CRF mRNA increased gradually and a significant elevation was first detected 60 min after the beginning of stress. Adrenalectomy augmented and dexamethasone pretreatment inhibited c-fos mRNA, CRF hnRNA, and mRNA induction after stress. The data suggest that stress-induced activation of neurons, CRF gene transcription, and CRF synthesis in the PVN are modulated by glucocorticoids.

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

Immunoreactive corticotropin-releasing factor in human plasma.

Plasma immunoreactive corticotropin-releasing factor (I-CRF) levels were determined by using a human CRF radioimmunoassay and an immunoaffinity procedure. The basal plasma I-CRF level in normal subjects was 6 +/- 0.5 pg/ml (mean +/- SD). We found that most plasma I-CRF levels were affected by stress, negative feedback, and circadian rhythm. Basal I-CRF levels were high in patients with Addison's disease, Nelson's syndrome, hypopituitarism stemming from pituitary macroadenoma, and CRF- and adrenocorticotropic hormone-producing tumors. A very low, but significant, amount of I-CRF was detected (1-3 pg/ml) in patients with Cushing's syndrome, in corticosteroid-treated patients, and in a patient with hypothalamic hypopituitarism. These results suggest that a major component of plasma I-CRF is of hypothalamic origin, however, other extrahypothalamic tissues cannot be ruled out as a minor source of plasma I-CRF.

Epidermal growth factor and transforming growth factor alpha induce ascitic fluid in mice.

Recent studies have suggested that pleural or peritoneal effusion associated with metastatic tumors is induced by some mediators produced by the tumor cells. We studied the ability of well-characterized peptide growth factors to produce ascites in mice. Peritoneal administration of epidermal growth factor (EGF, 10 to 40 micrograms/mouse/wk) or transforming growth factor alpha (TGF-alpha, 10 to 40 micrograms/mouse/wk) via osmotic minipumps resulted in formation of bloody ascites. The amount of ascites produced was dependent on the dose of growth factors. Vehicle alone or insulin-like growth factor I (40 micrograms/mouse/wk) was without effect. Indomethacin, a blocker of prostaglandin synthesis, significantly reduced the ascites accumulation induced by EGF, suggesting that prostaglandins are involved in ascites formation induced by EGF. Dexamethasone was also effective in attenuating the effect of EGF. Thus, it is possible that peritoneal effusion associated with disseminated tumors is, at least in part, due to EGF-like materials (most likely TGF-alpha) produced by tumor cells. The mechanism by which these peptides induce bloody ascites is not known for certain, but it may be due to the reported activity for neovascularization or increased vascular permeability.

Prolonged decrease of serum calcium concentration by murine gamma-interferon in hypercalcemic, human tumor (EC-GI)-bearing nude mice.

Murine gamma-interferon (MuIFN-gamma) is a potent inhibitor of bone resorption induced by interleukin 1 and parathyroid hormone-related protein in vitro. To investigate whether MuIFN-gamma is also effective in vivo, the cytokine was injected s.c. into hypercalcemic, tumor (EC-GI)-bearing nude mice, in which parathyroid hormone-related protein and interleukin 1 alpha are synergistically responsible for causing humoral hypercalcemia. When MuIFN-gamma was injected s.c. at a dose of 1 to 20 x 10(4) units for 5 days consecutively, serum calcium concentrations in the tumor-bearing mice decreased in a dose-dependent manner. The minimal effective dose was 5 x 10(4) units/mouse. Unlike calcitonin, which decreased the serum calcium concentration for only 1 to 2 days despite continuous daily injections, MuIFN-gamma decreased it for more than 7 days even after the injections had been stopped. Human gamma-interferon was completely ineffective. The decrease in serum calcium concentration was accompanied by a decrease in urinary calcium excretion. Histological examination of the femur revealed a decreased number of osteoclasts in the MuIFN-gamma-treated mice. Furthermore, MuIFN-gamma, when injected into nude mice or normal mice at a dose of 15 x 10(4) units for 3 days, almost completely abolished the formation of multinucleated osteoclast-like cells in vitro. These findings suggest that MuIFN-gamma suppresses the formation and maturation of osteoclasts and inhibits osteoclastic bone resorption, resulting in the prolonged decrease of serum calcium concentration seen in hypercalcemic, tumor-bearing nude mice. Therefore, bone resorption inhibitors like MuIFN-gamma, which ameliorate humoral hypercalcemia without an escape phenomenon, are potentially useful for the treatment of malignancy-associated hypercalcemia.

Potential of the histoculture drug-response assay to contribute to cancer patient survival.

The histoculture drug-response assay (HDRA) was recently evaluated in a retrospective clinical trial and was found to correlate to drug sensitivity, resistance, and patient survival. To further investigate the potential of HDRA to contribute to patient survival, 215 patients with gastric cancer from 45 medical centers were tested with the HDRA in a blinded study after resection of the primary lesion. One hundred sixty-eight patients received at least 20 mg/m2 of mitomycin C and a minimum of 30 g UFT, a mixture of tegafur and uracil at a molar ratio of 1:4, thereby making them eligible for the study. Of these cases 128 were evaluable by the HDRA. The evaluable patient tumors were tested by the HDRA with the [3H]thymidine incorporation end point measured by microautoradiography to be drug "sensitive" or "resistant." The in vitro conditions for distinguishing sensitivity and resistance that matched the response rates for historical controls for gastric carcinoma were 90% inhibition rate and 0.12 microgram/ml for mitomycin C and 70% inhibition rate and 1 microgram/ml for 5-fluorouracil, respectively. Most importantly in the blinded study, the overall and disease-free survival rates of the HDRA-sensitive group were found to be significantly higher than those of the HDRA-resistant group tested under the above conditions. The data further indicate the importance of three-dimensional tumor culture for obtaining accurate clinical information. The results demonstrate that the HDRA response correlates to patient survival, which suggests the potential of the HDRA to contribute to patient survival in gastric cancer when used prospectively.

Increased plasma endothelin in NIDDM patients with retinopathy.

OBJECTIVE: To elucidate the significance of ET in diabetic microvascular disease. RESEARCH DESIGN AND METHODS: We determined plasma levels of ir-ET-1 in 25 NIDDM patients without hypertension and/or renal dysfunction. RESULTS: The plasma levels of ir-ET-1 in NIDDM patients with simple (n = 8) and proliferative (n = 8) retinopathy were 0.58 +/- 0.04 pM and 0.60 +/- 0.04 pM, respectively, which were significantly higher than those in normal, nondiabetic subjects (0.24 +/- 0.02 pM [n = 31]) and NIDDM patients without retinopathy (0.30 +/- 0.05 pM [n = 9]). CONCLUSIONS: These results suggest that plasma ET-1 is related to diabetic microvascular disease.

Urinary excretion of parathyroid hormone-related protein fragments in patients with humoral hypercalcemia of malignancy and hypercalcemic tumor-bearing nude mice.

To investigate whether parathyroid hormone-related protein (PTHrP), a hypercalcemia-inducing factor responsible for malignancy-associated hypercalcemia (MAH), is excreted into urine of these patients, radioimmunoassay was established using antiserum specific for the C-terminal region of PTHrP-(127-141). Immunoreactive PTHrP (iPTHrP) was detected in the urine of all patients with MAH (n = 6) in whom nephrogenous cyclic AMP excretion was elevated. However, iPTHrP was not detected in the urine of normal subjects (n = 25) or hypercalcemic patients with primary hyperparathyroidism (n = 8). In normocalcemic patients with malignant disorders iPTHrP was not detected in the urine in most cases (24 of 25 patients) but was detectable in 1 of 25 patients. iPTHrP was also detected in the urine of hypercalcemic nude mice transplanted with PTHrP-producing tumors, but not in the urine of control and normocalcemic nude mice transplanted with PTHrP-nonproducing tumor. Furthermore, size-exclusion high-performance liquid chromatography revealed that the molecular weight of iPTHrP is about 2000-6000 daltons in the urine of patients as well as tumor-bearing nude mice. These data indicate that the fragments of the C-terminal region of PTHrP are excreted into the urine of patients with MAH and in a few normocalcemic patients with malignancies, suggesting that the measurement of iPTHrP in the urine is potentially useful in the differential diagnosis of hypercalcemia, particularly in differentiating humoral hypercalcemia of malignancy and primary hyperparathyroidism.

Passive immunization with anti-parathyroid hormone-related protein monoclonal antibody markedly prolongs survival time of hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors.

Malignancy-associated hypercalcemia is mainly caused by excessive production of parathyroid hormone-related protein (PTHrP) by the tumor. Using anti-PTHrP-(1-34) monoclonal murine antibody (anti-PTHrP MoAb), we studied whether repeated injection of the homologous antibody would continuously decrease the serum calcium concentration in hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors, leading to prolongation of their survival time. Daily SC injections of anti-PTHrP MoAb decreased the serum calcium concentration almost to within the normal range in nude mice bearing transplanted human PTHrP-producing tumors (T3M-1, EC-GI, PC-3, and FA-6) but not in a nude mouse bearing a transplanted parathyroid carcinoma. The antibody did not affect FA-6 tumor growth either in vitro or in vivo. Pancreatic carcinoma cells (FA-6), which caused the most severe hypercalcemia, were inoculated into 6-week-old nude mice. When severe hypercalcemia (approximately 19 mg/dl) had developed, daily SC injection of anti-PTHrP MoAb was started. Within 18 days of this time point, all untreated tumor-bearing mice (n = 10) died of hypercalcemia and cachexia, whereas all the treated mice (n = 10) showed an increase in body weight and survived for at least 25 days. Histologic examination of the treated mice revealed a marked decrease in osteoclastic bone resorption, without toxicologic findings in the kidney and liver. These results suggest that passive immunization against PTHrP can continuously ameliorate the hypercalcemia and markedly prolong the survival time of severely hypercalcemic, tumor-bearing mice. If a human monoclonal antibody against PTHrP-(1-34) could be developed, then passive immunization would be potentially one of the most effective therapies for patients with malignancy-associated hypercalcemia due to excessive production of PTHrP.

Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells.

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.

Changes in neoplastic cell features and sensitivity to mitotane during mitotane-induced remission in a patient with recurrent, metastatic adrenocortical carcinoma.

A 58-year-old man had adrenocortical carcinoma in the right adrenal gland. The tumour secreted excessive cortisol and dehydroepiandrosterone-sulphate (DHEA-S), and had invaded the right hepatic lobe and vena cava. Eleven months after surgical tumour resection, the serum DHEA-S levels again increased. Local tumour recurrence and a metastasis was found in the lung. Eleven months after surgery chemotherapy with mitotane (o,p'-DDD) was initiated. Twelve weeks of mitotane reduced serum DHEA-S levels and caused these tumours to disappear. The patient was then treated with low-dose mitotane (1.5-2.0 g/day) for 2 years. Serum levels of mitotane remained at less than 10 microg/ml. Although such low serum levels of mitotane and delayed initiation of mitotane after surgery have been proposed to weaken the antineoplastic effect of mitotane, the patient had a remission for 2 years. However, there was then local re-recurrence with an increase in serum DHEA-S and death 4 months later. The histological features of neoplastic cells were quite different comparing tumour resected at surgery and tumour at autopsy. The latter had more frequent mitotic nuclei. This tumour was initially sensitive to mitotane, but later became insensitive.

Effect of antiserum to rat growth hormone (GH)-releasing factor on physiological GH secretion in the female rat.

To further study the physiological role of GH-releasing factor (GRF), we examined the effect of antiserum to rat hypothalamic GRF on spontaneous GH secretion in the normal female rat. Two groups of six conscious female rats were passively immunized with either nonimmune rabbit serum (NRS) or antirat GRF serum via a chronic indwelling atrial catheter. The secretory profiles of GH were observed by collecting blood samples at 15-min intervals for 1 h before and 4 h after administration. The NRS-treated rats showed a characteristic female pattern of spontaneous GH secretion. GH pulses were of low amplitude (mean +/- SEM, 26.8 +/- 2.4 ng/ml) and occurred irregularly at a frequency of 4.2 +/- 0.2/5 h, while interpeak through levels of GH were relatively high, with nadir values of 8.6 +/- 0.7 ng/ml. Synthetic rat GRF, given iv at a dose of 1 microgram/kg BW after the last blood sampling, stimulated GH release to a peak level of 153 +/- 37 ng/ml in the control rats. Administration of GRF antiserum caused a profound suppression of both pulse and trough components of GH secretion. This effect occurred rapidly, within 15 min after injection of antiserum, and GH secretion decreased uniformly to very low levels (3.4 +/- 0.1 ng/ml), with little or no fluctuation throughout the observation period. GRF antiserum also abolished the synthetic rat GRF-induced GH release, indicating sufficient potency of immunoneutralization. These results demonstrate that both GH pulses and troughs are dependent upon hypothalamic GRF in normal female rats, thereby substantiating earlier observations in male rats which demonstrated the physiological role of GRF in GH secretion.

Stimulation of renal deoxyribonucleic acid synthesis by a pituitary-derived renotropin and its inhibition by testosterone and thyroxine.

Kidney slices were obtained from castrated-hypophysectomized male rats treated with a single injection of several different gonadotropin preparations (two ovine LH fractions, one bovine LH fraction, and one hCG fraction) or vehicle, then incubated in a buffer containing [3H]thymidine. Only one of the above, an ovine LH preparation, increased [3H]thymidine incorporation into renal DNA, with a peak occurring 8-10 h after injection and therefore termed renotropin. However, in hypophysectomized rats with intact testes, such an effect was not observed. Furthermore, while testosterone propionate alone did not alter basal incorporation in castrated-hypophysectomized rats, it abolished the incorporation that was stimulated by renotropin. These results suggest that androgens, whether of testicular origin or exogenously administered, suppress the increased incorporation of [3H]thymidine stimulated by renotropin. This antagonistic effect of androgen was also observed with T4, but to a lesser degree. Our findings confirm the presence of renotropin, which could not be attributed to other known pituitary hormones, and suggest that there is a complex interaction between this factor and two other renal growth-promoting hormones, testosterone and T4.

Early induction of c-fos precedes increased expression of corticotropin-releasing factor messenger ribonucleic acid in the paraventricular nucleus after immobilization stress.

CRF plays a role in coordinating endocrine, physiological, and behavioral responses to stressful stimuli. Several kinds of stressors have been reported to induce an increase in CRF mRNA expression in the paraventricular nucleus of the hypothalamus (PVN). Recently, the expression of c-fos mRNA has shown promise as a useful tool for metabolic mapping at the cellular level, because various types of stimulation induce c-fos mRNA expression in specific neuron populations in various brain regions. The aim of the present study is to clarify a possible anatomical-temporal correlation between the early induction of c-fos and the enhanced expression of CRF mRNA after stress. Wistar male rats were exposed to immobilization stress for 60 min and killed before and 15, 30, 60, 90, 120, and 180 min after the beginning of immobilization. In situ hybridization was performed by hybridizing sections with 35S-labeled prepro-CRF and c-fos cRNA probes. Relative levels of CRF and c-fos mRNA were compared by estimating the number of grains over the PVN in emulsion-dipped autoradiograms. Rapid induction (within 15 min) of c-fos mRNA was noted in the parvocellular division of the PVN after immobilization stress. The level of c-fos mRNA peaked at 30 min, then gradually declined to the control level within 90 min after the beginning of stress [the number of grains over the PVN: control, 326 +/- 180; 15 min, 2091 +/- 680 (P less than 0.05 vs. control); 30 min, 3385 +/- 239 (P less than 0.05 vs. control)]. The distribution of c-fos mRNA was almost identical to that of CRF mRNA in the PVN. On the other hand, the time course of CRF mRNA induction was delayed to the c-fos mRNA expression. A significant increase in CRF mRNA levels was noted only 120 and 180 min after stress [the number of grains over PVN: control, 3868 +/- 221; 120 min, 5957 +/- 677 (P less than 0.05 vs. control); 180 min, 6600 +/- 450 (P less than 0.05 vs. control)]. The results demonstrate that increased expression of CRF mRNA is preceded by c-fos mRNA induction in the PVN after stress suggesting a role of c-fos in the activation of CRF gene expression.

Anabolic effects of 1,25-dihydroxyvitamin D3 on osteoblasts are enhanced by vascular endothelial growth factor produced by osteoblasts and by growth factors produced by endothelial cells.

Human osteoblast-like cells (HOB) produce vascular endothelial growth factor (VEGF), the steady state level of which is stimulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. As osteoblasts and endothelial cells are proximally located in skeletal tissue, we investigated the anabolic effects of 1,25-(OH)2D3 and VEGF on HOB cocultured with endothelial cells. When HOB with high alkaline phosphatase (Al-P) activity and human umbilical vein endothelial cells (HUVEC) with little activity were cultured together, Al-P activity increased, accompanied by an increase in cell number. When HOB and HUVEC were cultured separately, 1,25-(OH)2D3 did not directly stimulate [3H]thymidine incorporation into HUVEC, but stimulated it in the presence of HOB. VEGF did not directly stimulate the Al-P activity of HOB but stimulated it in the presence of HUVEC. The conditioned medium of HOB stimulated the proliferation of HUVEC, and this was partially blocked by anti-VEGF antibody. Conversely, the conditioned medium of HUVEC increased Al-P activity and [3H]thymidine incorporation into HOB, and this was partially blocked by antiinsulin-like growth factor I antibody and BQ-123, a specific antagonist of the endothelin-1 (ET-1) receptor. 1,25-(OH)2D3 stimulated the release of VEGF and ET-1 from HOB and HUVEC, respectively. Furthermore, the 1,25-(OH)2D3-induced release of VEGF was enhanced in HOB cocultured with HUVEC. A quantitative reverse transcription-PCR study revealed that genes for VEGF receptors (Flt-1 and KDR) were expressed in HUVEC, but not in HOB, and that 1,25-(OH)2D3 increased the levels of expression of VEGF receptor genes in endothelial cells only when cocultured with HOB. In summary, we demonstrated that 1,25-(OH)2D3 exerts an anabolic effect on osteoblasts by enhancing their production of VEGF, which stimulates its receptors on endothelial cells, followed by increased production of osteotropic growth factors, such as insulin-like growth factor I and ET-1. These in vitro findings suggest that the VEGF/VEGF receptor system may be involved in both bone formation and bone remodeling in vivo.