HL60 cells differentiate to monocytes or neutrophils in response to 1 alpha,25(OH)2-vitamin D3 (D3) and retinoids respectively. D3 and retinoid actions converge since their receptors (VDR, RAR) heterodimerise with a common partner, RXR, which also interacts with thyroid hormone (T3) receptors (T3R). HL60 cells were treated with combinations of D3 and retinoids to induce differentiation and to investigate whether increased VDR or RAR expression correlated with monocyte or neutrophil differentiation and whether altered receptor concentrations affected DNA-binding specificity. As assessed by Western blotting, VDR and RXR expression was unchanged in monocytes relative to controls but levels of RAR and T3R were reduced. In contrast, only VDR expression was reduced in neutrophils. T3 did not promote differentiation or influence its induction by D3 or retinoids and did not affect expression of any receptor. Gel mobility-shift analysis revealed that nuclear extracts from undifferentiated cells, monocytes and neutrophils interacted differently with VDRE-, RARE- and RXRE-binding sites. Monocyte nuclear protein/DNA complexes contain readily detectable VDR and RXR whereas neutrophil complexes contain RAR and RXR. Thus hormone-induced changes in receptor stoichiometry favour either VDR/RXR or RAR/RXR heterodimerisation and correlate with hormone-induced differentiation of HL60 cells to monocytes or neutrophils respectively.

Goitrogenesis is accompanied by hyperplasia and hypertrophy and involves tissue remodelling and angiogenesis. During the involution of the goitre there must be removal of this increased thyroid volume, in addition to further remodelling, which may involve apoptosis. We investigated apoptosis in the involuting rat thyroid using male Fisher rats that were on a goitrogenic regimen for 14 days and then returned to a normal diet. Thyroid weights increased fourfold with the goitrogenic regimen. During involution, the largest decrease in weight was between day 2 and day 4 after withdrawal of treatment. After 34 days of involution, the thyroid weight plateaued, but had not returned to control values. High levels of Bcl-2 immunoreactivity were observed in normal and goitrous rat thyroids. These high levels were significantly reduced at 2 days of involution, after which high levels of Bcl-2 immunoreactivity returned. In situ end-labelling of apoptotic cells showed that there was an increase in the number of cells undergoing DNA fragmentation during goitrogenesis (1.0+/-0.8 cells/100 cells, n=9) compared with controls, in which no positive staining was observed. After 2 days of goitrogen withdrawal, there was a further fourfold increase in the number of in situ end-labelled cells (day 16: 4.1+/-1.7, n=9). Numbers of positive cells returned to low levels after 4 days of involution (day 18: 0.3+/-0.8, n=9). Using antiserum to apoptosis-specific protein, we found increased immunoreactivity during goitrogenesis and after 2 days of involution that was localised predominantly with the stromal and vascular tissue at both time points. The data show that rapid downregulation of Bcl-2 accompanies thyroid involution, which involves increased levels of apoptosis within the stromal compartment.

Studies in vitro and in vivo have shown that corticosteroids play an important role in bone physiology and pathophysiology. It is now established that corticosteroid hormone action is regulated, in part, at the pre-receptor level through the expression of isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which are responsible for the interconversion of hormonally active cortisol to cortisone. In this report we demonstrate 11beta-HSD activity in human osteoblast (OB) cells. Osteosarcoma-derived OB cell lines TE-85, MG-63 and SaOS-2 and fibrosarcoma Hs913T cells express the type 2 isoform of 11beta-HSD, as determined by reverse transcription polymerase chain reaction (RT-PCR) and specific enzyme assays. Enzyme activity was shown to be strictly NAD dependent with a Km of approximately 71 nM; 11beta-HSD type 1 mRNA expression and enzyme activity were not detected. All four cell lines expressed mRNA for the glucocorticoid receptor (GR) and mineralocorticoid receptor, but specific binding was only detectable with radiolabelled dexamethasone (Kd=10 nM) and not aldosterone. MG-63 cells had two to three times more GR than the other OB cells, which correlated with the higher levels of 11beta-HSD 2 activity in these cells. In contrast to the osteosarcoma cell studies, RT-PCR analysis of primary cultures of human OB cells revealed the presence of mRNA for 11beta-HSD 1 as well as 11beta-HSD 2. However, enzyme activity in these cells remained predominantly oxidative, i.e. inactivation of cortisol to cortisone (147 pmol/h per mg protein at 500 nM cortisol) was greater than cortisone to cortisol (10.3 pmol/h per mg protein at 250 nM cortisone). Data from normal human OB and osteosarcoma cells demonstrate the presence of an endogenous mechanism for inactivation of glucocorticoids in OB cells. We postulate that expression of the type 1 and type 2 isoforms of 11beta-HSD in human bone plays an important role in normal bone homeostasis, and may be implicated in the pathogenesis of steroid-induced osteoporosis.

ABSTRACT

We have reported previously the effect of thyroid status in vivo on pituitary cytoplasmic concentrations of messenger RNA (mRNA) encoding the thyrotrophin (TSH) β-subunit (Franklyn, Lynam, Docherty et al, 1985). Studies in vitro of the regulation of TSH β gene transcription have been confined to thyrotrophic tumour cells. We now report the demonstration of TSH β-subunit mRNA in non-tumorous rat pituitary cells in primary culture. Treatment of cells with thyrotrophin-releasing hormone (TRH) and with forskolin resulted in a marked increase in cellular concentration of TSH β-mRNA. These results suggest that TRH exerts a direct effect on the pretranslational events involved in TSH synthesis and further that the adenylate cyclase system may be involved in the regulation of synthesis. We have thus described a novel system for the study of TSH β-subunit gene expression in normal rat pituitary cells in vitro.