A possible role for a low molecular weight peptide in regulation of testosterone production by rat Leydig cells

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Percoll purified Leydig cell proteins from 20- and 120-day-old rats revealed a significant decrease in a low molecular weight peptide in the adult rats. Administration of human chorionic gonadotropin to immature rats resulted in a decrease in the low molecular weight peptide along with increase in testosterone production. Modulation of the peptide by human chorionic gonadotropin could be confirmed by Western blotting. The presence of a similar peptide could be detected by Western blotting in testes of immature mouse, hamster, guinea pig but not in adrenal, placenta and corpus luteum. Administration of testosterone propionate which is known to inhibit the pituitary luteinizing hormone levels in adult rats resulted in an increase in the low molecular weight peptide, as checked by Western blotting. It is suggested that this peptide may have a role in regulation of acquisition of responsiveness to luteinizing hormone by immature rat Leydig cells.

Regulation and Function of GATA Transcription Factors in Adrenocortical Tumors and Granulosa Cells

Transcription factors play a key role in tumor development, in which dysfunction of genes regulating tissue growth and differentiation is a central phenomenon. The GATA family of transcription factors consists of six members that bind to a consensus DNA sequence (A/T)GATA(A/G) in gene promoters and enhancers. The two GATA factors expressed in the adrenal cortex are GATA-4 and GATA-6. In both mice and humans, GATA-4 can be detected only during the fetal period, whereas GATA-6 expression is abundant both throughout development and in the adult. It is already established that GATA factors are important in both normal development and tumorigenesis of several endocrine organs, and expression of GATA-4 and GATA-6 is detected in adrenocortical tumors. The aim of this study was to elucidate the function of these factors in adrenocortical tumor growth. In embryonal development, the adrenocortical cells arise and differentiate from a common pool with gonadal steroidogenic cells, the urogenital ridge. As the adult adrenal cortex undergoes constant renewal, it is hypothesized that undifferentiated adrenocortical progenitor cells reside adjacent to the adrenal capsule and give rise to daughter cells that differentiate and migrate centripetally. A diverse array of hormones controls the differentiation, growth and survival of steroidogenic cells in the adrenal gland and the gonads. Factors such as luteinizing hormone and inhibins, traditionally associated with gonadal steroidogenic cells, can also influence the function of adrenocortical cells in physiological and pathophysiological states. Certain inbred strains of mice develop subcapsular adrenocortical tumors in response to gonadectomy. In this study, we found that these tumors express GATA-4, normally absent from the adult adrenal cortex, while GATA-6 expression is downregulated. Gonadal markers such as luteinizing hormone receptor, anti-Müllerian hormone and P450c17 are also expressed in the neoplastic cells, and the tumors produce gonadal hormones. The tumor cells have lost the expression of melanocortin-2 receptor and the CYP enzymes necessary for the synthesis of corticosterone and aldosterone. By way of xenograft studies utilizing NU/J nude mice, we confirmed that chronic gonadotropin elevation is sufficient to induce adrenocortical tumorigenesis in susceptible inbred strains. Collectively, these studies suggest that subcapsular adrenocortical progenitor cells can, under certain conditions, adopt a gonadal fate. We studied the molecular mechanisms involved in gene regulation in endocrine cells in order to elucidate the role of GATA factors in endocrine tissues. Ovarian granulosa cells express both GATA-4 and GATA-6, and the TGF-β signaling pathway is active in these cells. Inhibin-α is both a target gene for, and an atypical or antagonistic member of the TGF-β growth factor superfamily. In this study, we show that GATA-4 is required for TGF-β-mediated inhibin-α promoter activation in granulosa cells, and that GATA-4 physically interacts with Smad3, a TGF-β downstream protein. Apart from the regulation of steroidogenesis and other events in normal tissues, TGF-β signaling is implicated in tumors of multiple organs, including the adrenal cortex. Another signaling pathway found often to be aberrantly active in adrenocortical tumors is the Wnt pathway. As both of these pathways regulate the expression of inhibin-α, a transcriptional target for GATA-4 and GATA-6, we wanted to investigate whether GATA factors are associated with the components of these signaling cascades in human adrenocortical tumors. We found that the expression of Wnt co-receptors LRP5 and LRP6, Smad3, GATA-6 and SF-1 was diminished in adrenocortical carcinomas with poor outcome. All of these factors drive inhibin-α expression, and their expression in adrenocortical tumors correlated with that of inhibin-α. The results support a tumor suppressor role previously suggested for inhibin-α in the mouse adrenal cortex, and offer putative pathways associated with adrenocortical tumor aggressiveness. Unraveling the role of GATA factors and associated molecules in human and mouse adrenocortical tumors could ultimately contribute to the development of diagnostic tools and future therapies for these diseases.

A new method for modulating the activity of parvalbumin-positive neurons and cerebellar Purkinje cells in vivo

Neurons can be divided into various classes according to their location, morphology, neurochemical identity and electrical properties. They form complex interconnected networks with precise roles for each cell type. GABAergic neurons expressing the calcium-binding protein parvalbumin (Pv) are mainly interneurons, which serve a coordinating function. Pv-cells modulate the activity of principal cells with high temporal precision. Abnormalities of Pv-interneuron activity in cortical areas have been linked to neuropsychiatric illnesses such as schizophrenia. Cerebellar Purkinje cells are known to be central to motor learning. They are the sole output from the layered cerebellar cortex to deep cerebellar nuclei. There are still many open questions about the precise role of Pv-neurons and Purkinje cells, many of which could be answered if one could achieve rapid, reversible cell-type specific modulation of the activity of these neurons and observe the subsequent changes at the whole-animal level. The aim of these studies was to develop a novel method for the modulation of Pv-neurons and Purkinje cells in vivo and to use this method to investigate the significance of inhibition in these neuronal types with a variety of behavioral experiments in addition to tissue autoradiography, electrophysiology and immunohistochemistry. The GABA(A) receptor γ2 subunit was ablated from Pv-neurons and Purkinje cells in four separate mouse lines. Pv-Δγ2 mice had wide-ranging behavioral alterations and increased GABA-insensitive binding indicative of an altered GABA(A) receptor composition, particularly in midbrain areas. PC-Δγ2 mice experienced little or no motor impairment despite the lack of inhibition in Purkinje cells. In Pv-Δγ2-partial rescue mice, a reversal of motor and cognitive deficits was observed in addition to restoration of the wild-type γ2F77 subunit to the reticular nucleus of thalamus and the cerebellar molecular layer. In PC-Δγ2-swap mice, zolpidem sensitivity was restored to Purkinje cells and the administration of systemic zolpidem evoked a transient motor impairment. On the basis of these results, it is concluded that this new method of cell-type specific modulation is a feasible way to modulate the activity of selected neuronal types. The importance of Purkinje cells to motor control supports previous studies, and the crucial involvement of Pv-neurons in a range of behavioral modalities is confirmed.

Enhanced photodynamic effect of cobalt(III) dipyridophenazine complex on thyrotropin receptor expressing HEK293 cells

Ternary cobalt(III) complexes CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at similar to 690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K-d value of 81 nM and from the photocleavage of hTSHR-ECD.