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ABSTRACT

The present studies have demonstrated the production of transforming growth factor-β1 (TGF-β1) by porcine thyroid follicular cells (TFCs) maintained in vitro as subconfluent monolayers, and have confirmed a stimulatory effect of iodide on thyroidal TGF-β1 mRNA and peptide release. RNA extracted from TFCs maintained in the absence of iodide contained a 2·5 kb transcript which hybridized specifically with a cDNA probe for human TGF-β1, and which showed an approximate doubling in intensity in cells exposed to 10 μmol NaI/l. In the presence of the anti-thyroid thionamide drug methimazole (MMI; 1 mmol/l), the action of iodide on TGF-β1 mRNA was attenuated, although MMI alone had no effect on the control level of TGF-β1 mRNA. The TGF-β1 peptide content of TFC-conditioned media (TFC-CM) was assessed using the fetal mink lung cell line Mv1 Lu, in which activated TGF-β1 specifically suppresses trichloroacetic acid-precipitable [methyl-³H]thymidine incorporation. Newly conditioned TFC-CM stimulated [methyl-³H]thymidine incorporation into Mv1Lu cells, but after heat treatment to inactivate growth stimulators and activate the latent TGF-β1 component this medium inhibited [methyl-³H]thymidine incorporation. This inhibitory effect was prevented by immunoadsorption of TFC-CM with a TGF-β1-neutralizing antiserum, confirming the specificity of the inhibitory response. The inhibitory activity of TFC-CM was increased when the TFCs were preincubated with 10 μmol NaI/l, and lost when TFCs were exposed to MMI. In conclusion, TFCs produce TGF-β1 mRNA and TGF-β1 peptide, which are both increased by iodide treatment in vitro. The anti-thyroid effects of MMI may, at least in part, be mediated by a decrease in TFC-derived TGF-β1 production.

ABSTRACT

Pit-1, a member of the POU family of homeodomain transcription factors, activates prolactin and GH gene expression but also has a role in pituitary cell differentiation and proliferation. Expression of Pit-1 may therefore be of central importance in the function and phenotype of human pituitary adenomas. We have found evidence that, in addition to Pit-1 mRNA, Pit-1-like immunoreactivity and DNA-binding activity are readily detectable in a series of human pituitary adenomas. Gel mobility shift assays using adenoma protein extracts with two Pit-1-binding sites from the human prolactin gene promoter demonstrated the formation of several DNA sequence-specific protein—DNA complexes; some of these could be accounted for by Oct-1-binding activity. Pit-1 activity was anticipated in prolactin- and GH-secreting adenomas, but was also detected in a proportion of endocrine-inactive (non-secreting) adenomas that did not express Pit-1 target genes.

The data demonstrate the presence of Pit-1 in a range of pituitary adenomas. Different adenomas generated slightly differing patterns of DNA-binding activity, though Pit-1 mRNA and protein size appeared normal in all tumours so far examined.

ABSTRACT

Stimulation of prolactin secretion by TRH probably involves mobilization of intracellular calcium to a greater extent than calcium influx, but less is known about the possible calcium-dependent mechanisms which may control prolactin gene transcription. We have studied this question in the rat pituitary tumour GH₃ cell line by measuring prolactin mRNA accumulation by a cytoplasmic dot hybridization assay. Cobalt chloride, an intracellularly acting calcium antagonist, caused marked dose-dependent reductions in prolactin release and mRNA concentrations, whereas the calcium channel-blocking agent verapamil had no effect on prolactin release and had smaller effects on prolactin mRNA. Cobalt chloride abolished the stimulatory effect of TRH on prolactin mRNA levels, while verapamil caused only moderate inhibition. Growth hormone mRNA levels in the same cells were not significantly affected by TRH or verapamil, and only marginally reduced by cobalt. These data suggest that, as for prolactin release from normal rat pituitary lactotrophs, prolactin mRNA accumulation in GH₃ cells appears to have a requirement for intracellular calcium which is only partly dependent upon calcium influx.

ABSTRACT

IGF-I mRNA has been demonstrated in testicular tissue and, more recently, localized specifically to Leydig cells. This study investigated the expression of IGF-I and side-chain cleavage enzyme (SCC) mRNA in two preparations of rat interstitial testicular cells which were separated by buoyant density into Leydig cell-enriched and -depleted fractions. RNA was prepared from interstitial cells obtained from the testes of untreated adult and immature rats and adult rats treated with human chorionic gonadotrophin (hCG) or ethane dimethanesulphonate (EDS; to destroy Leydig cells). IGF-I mRNA was detected in all samples, with five major transcripts ranging from 7·5 to 0·6 kb. Leydig cells (3β-hydroxysteroid dehydrogenase-positive and sensitive to EDS) expressed abundant IGF-I and SCC mRNAs, and levels of both were increased following hCG treatment. However, in addition, IGF-I mRNA which was derived from non-Leydig interstitial cells was detected, in the complete absence of SCC message, either in the more buoyant interstitial cells or in both interstitial cell fractions following the destruction of Leydig cells by EDS treatment. IGF-I expression in the Leydig cell-depleted cell fraction was also increased by hCG treatment, and it is therefore suggested that at least part of this non-Leydig interstitial cell IGF-I mRNA originates in Leydig cell precursors. In conclusion, Leydig cells are not the sole origin of IGF-I mRNA in the testis, and the non-Leydig cell expression may be an important component of testicular IGF-I production.

ABSTRACT

In the normal pituitary, glucocorticoids are the principal negative regulator of the pro-opiomelanocortin (POMC) gene which gives rise to the biologically active peptides ACTH and β-endorphin. In Cushing's syndrome, ACTH-secreting pituitary tumours show a degree of glucocorticoid resistance, whilst ACTH-secreting extra-pituitary tumours have an even greater resistance to glucocorticoid excess. In an attempt to understand the mechanism of this phenomenon, we have compared the effects of glucocorticoids on POMC mRNA and peptide secretion in human and mouse corticotroph adenoma cells and in small cell lung carcinoma (SCLC) cells. ACTH precursor peptides were inhibited within 24 h by 25–50 nm hydrocortisone in primary cultures from a human corticotroph adenoma. In the mouse corticotroph adenoma cell line (AtT20), inhibition of both ACTH precursors and ACTH was not observed after 24 h but, by 10 days, glucocorticoids suppressed peptide levels with a concentration causing 50% inhibition of 50 nm hydrocortisone and maximal inhibition at 500 nm hydrocortisone. In marked contrast, there was no response to 500 nm hydrocortisone in the five SCLC cell lines (COR L103, COR L42, COR L24, COR L31, DMS 79) all of which secrete ACTH precursors. However, two of the five SCLC cell lines (COR L31 and DMS 79) were responsive to 1000 nm hydrocortisone. POMC mRNA, quantitated by slot-blot analysis, gave similar results for the five SCLC cell lines, implying that the abnormality may occur at the level of gene expression. When one of the three resistant cell lines (COR L103) was incubated with 2000 nm hydrocortisone or 2000 nm dexamethasone a clear suppression of precursor peptides and POMC mRNA was observed. This suggests that the resistance to glucocorticoid inhibition is relative rather than absolute, implying that the normal mechanism is functioning but impaired. Furthermore, there is at least a 20-fold difference in the responsiveness to glucocorticoid inhibition between pituitary and extra-pituitary tumour cells in vitro, which may signify a difference in the underlying mechanism in these two cell types.

ABSTRACT

A transcriptional enhancer which has a consensus binding sequence for transcription enhancer factor-1 (TEF-1) has been found 3′ of the hPL₃ gene. We examined whether TEF-1 is expressed by the human placenta and whether such expression is co-ordinated with that of human placental lactogen (hPL). Probing Northern blots of total RNA from first trimester and term placenta, the choriocarcinoma-derived cell line JAr and primary cultured cytotrophoblast cells with a cDNA for TEF-1 revealed transcripts of 12–13 kb and 3–4 kb. The level of TEF-1 expression was the same in first trimester as compared with term placenta and in undifferentiated JAr as compared with differentiated cytotrophoblast cells. hPL expression was tenfold higher in term compared with first trimester placenta and, whilst detectable in cytotrophoblast cells, was undetectable in JAr cells. These data show that TEF-1 is expressed by the placenta but is not co-ordinated with hPL expression.