ABSTRACT

We have previously demonstrated that retinoic acid (RA) as well as thyroid hormone stimulates GH gene expression. To clarify the relationship between the action of RA and thyroid hormone, pituitary-specific gene expression was investigated further in rat pituitary cells.

Rat clonal pituitary cells, GH₃, were treated with RA with or without tri-iodothyronine (T₃) for up to 3 days. After treatment with 10–1000 nmol RA/1 with or without 0·1–10 nmol T₃/1, medium was collected for radioimmunoassay and cells were subjected to RNA extraction, and GH and prolactin gene expression was analysed using ³²P-labelled rat GH and rat prolactin cDNA probes respectively. The data demonstrated the dose–responsive manner of the stimulatory effects of RA and T₃ on GH secretion with T₃-depleted media. The action of RA was additive to that of T₃ for GH secretion when maximum effective doses of RA or T₃ were used. Using dot blot and Northern gel analysis, it was shown that RA increased GH mRNA levels in T₃-depleted media, and that this action of RA was additive to that of T₃ on the induction of GH mRNA levels. In contrast, neither RA nor T₃ stimulated the secretion of prolactin and prolactin mRNA levels in these cells.

Our results indicate that RA stimulates GH mRNA increment and GH secretion in T₃-depleted media, and that the stimulatory effect of RA is additive to the maximum effective dose of T₃.

Journal of Endocrinology (1990) 125, 251–256

Abstract

The in vivo responsiveness of thyroid glands to TSH at various ages in novel 'growth-retarded' (grt/grt) mice derived from Snell's dwarf (DW/J) mice and in their normal counterparts were analysed by determining serum T₄ concentrations before and after the administration of exogenous TSH. The serum T₄ concentration in normal mice increased in response to TSH at 2, 4 and 12 weeks of age but not at 1 week of age. A transient augmentation of such thyroidal responsiveness to TSH was apparent in normal mice at 2 weeks of age, when the serum T₄ level exhibits a peak and the pubertal growth of mice starts. In contrast to normal mice, at any age examined from 2 to 12 weeks after birth, exogenous TSH did not influence serum T₄ concentrations in the grt/grt mice at all. On the other hand, serum TSH concentrations in young grt/grt mice were highly elevated compared with those in normal mice and they were normalized by a 2–3 week's treatment with T₃. Morphological studies demonstrated degenerated thyroid glands in the grt/grt mice. These results suggest that the severe hypothyroidism and consequent growth retardation in growth-retarded mice are due to impairment of the thyroid glands of the mutant mice in producing and/or secreting thyroid hormones in response to TSH.

Journal of Endocrinology (1995) 144, 209–214

It has been recognized that tissue-specific growth factors and angiogenic factors play important roles in the growth of tumors and in the tissue-repair system. In uterine myometrial smooth muscle cells, it has also been reported that the platelet-derived growth factor (PDGF) binds to PDGF receptors and stimulates proliferation. In this paper, we examine whether or not PDGF is able to stimulate production of vascular endothelial growth factor (VEGF) in cultured human myometrial smooth muscle cells. PDGF treatment enhanced immunoreactive VEGF production as well as cell proliferation. Production of VEGF121 and VEGF165 in the cells was detected by reverse transcription-polymerase chain reaction analysis, but the PDGF treatment did not change the ratio of VEGF165 to VEGF121. The effect of PDGF on cell proliferation leveled off at 10 ng/ml, whereas its effect on VEGF production continued to increase linearly at concentrations above 10 ng/ml. Upon treatment of the cells with antibody against VEGF, the cell proliferation increased linearly even at PDGF concentrations above 10 ng/ml. The enhanced [3H]thymidine incorporation by PDGF was abolished by either mitogen-activated protein kinase kinase (MAPKK) inhibitor or protein kinase C (PKC) inhibitor. In contrast, VEGF production was abolished by MAPKK inhibitor, but not by PKC inhibitor. These results indicate that PDGF stimulates both cell proliferation and VEGF production in partly different signal pathways, and thus PDGF might play a role in the physiology and pathology of the myometrium.

Osteoclasts are formed from hematopoietic precursors via cell-cell fusion. We have previously reported that mannose residues are expressed on the outer membranes of monocytes during osteoclast differentiation. In the present study, we have attempted to demonstrate the pattern of expression levels of terminal high-mannose type oligosaccharide and to show that the mannose receptor is expressed on osteoclast precursor cells. Osteoclasts were formed using three different systems, namely mouse bone marrow cell culture, co-culture of mouse spleen cells with stromal cells, and RAW264.7 cell cultures. During osteoclast differentiation, the expression of terminal high-mannose type oligosaccharide gradually increased and then peaked at the stage of fusion in all three systems. Expression of the mannose receptor gradually increased during osteoclast differentiation in bone marrow cells and the co-culture system. In contrast, that in RAW264.7 cells had already been detected in the absence of the soluble receptor activator of NF-kappaB ligand and did not change during osteoclast differentiation. To ascertain whether expression of high-mannose type oligosaccharide is involved in tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation, glycosidase inhibitors were used on RAW264.7 cell culture. Castanospermine, an inhibitor of glucosidase I, inhibited the TRAP-positive MNCs, and deoxymannojirimycin, an inhibitor of alpha-mannosidase I, increased the TRAP-positive MNC formation. These results indicate that the binding of terminal high-mannose and mannose receptor is important for the process of cellular fusion in osteoclast formation.

ABSTRACT

Neurocalcin (molecular weight 23 000 and 24 000) is a Ca²⁺-binding protein with three putative Ca²⁺-binding domains and is present in large amounts in nervous tissues. Neurocalcin isoproteins separated by C₁₈ reverse-phase column chromatography are insoluble in buffer solution and it is impossible to determine the dissociation constant of neurocalcin with Ca²⁺. To overcome this difficulty, recombinant neurocalcin was synthesized, based on one of the cDNAs of the neurocalcin isoproteins. Stoichiometric titration experiments, using recombinant neurocalcin, indicated that this protein bound 2 mol Ca²⁺/mol protein and that the apparent dissociation constant for Ca²⁺ was 2·2 μmol/l, suggesting that neurocalcin plays a physiological role in cellular function. Immunoblotting showed that neurocalcin is present in the bovine adrenal gland in addition to the nervous tissues. Neurocalcin, identified by immunoblotting, was purified from the bovine adrenal gland. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of neurocalcin from the bovine brain showed 23 kDa and 24 kDa double bands, while SDS-PAGE of neurocalcin from the adrenal gland showed a single band of apparently 24 kDa, suggesting that the expression of neurocalcin isoproteins differs from tissue to tissue. The content of neurocalcin in the adrenal gland was 10 μg protein/100 g wet tissue. Immunohistochemical analysis showed the occurrence of neurocalcin in zona glomerulosa and adrenal medulla but not in zona fasciculata or zona reticularis. The restricted localization of neurocalcin in the adrenal gland suggests that a similar Ca²⁺ signal pathway may be present in zona glomerulosa and the adrenal medulla.

Journal of Endocrinology (1993) 138, 283–290