SKOV3 cells containing a truncated ARID1a protein have a restricted genome-wide response to glucocorticoids.

AT-rich interacting domain subunit 1a (ARID1a) is an essential SWI/SNF component frequently mutated in human cancers. ARID1a mutations have also been associated with glucocorticoid resistance, potentially related to the well-established role of the SWI/SNF complex in glucocorticoid target gene regulation. Glucocorticoids are steroid hormones important for regulating many physiological processes through the activation of the glucocorticoid receptor (GR). As GR interacts directly with ARID1a, we hypothesized that a truncating ARID mutation would interfere with GR-dependent gene regulation. Using high throughput RNA sequencing (RNA-SEQ) we show a restricted glucocorticoid response in SKOV3 cells, which contain an inactivating ARID1a mutation. We also show a lack of GR binding at the GR-dependent regulatory site in the Period 1 gene, which has previously been shown to require chromatin remodelling. Taken together, our data suggests that ARID1a may be required for regulation of a subset of glucocorticoid responsive genes. In the case of SKOV3 cells, in which ARID1a is mutated, glucocorticoid-dependent transcriptional regulation of these genes is significantly impaired.

Glucocorticoid ultradian rhythmicity directs cyclical gene pulsing of the clock gene period 1 in rat hippocampus.

In vivo glucocorticoid (GC) secretion exhibits a distinctive ultradian rhythmicity. The lipophilic hormone can rapidly diffuse into cells, although only the pulse peak is of sufficient amplitude to activate the low affinity glucocorticoid receptor (GR). Discrete pulses readily access brain regions such as the hippocampus where GR expression is enriched and known to regulate neuronal function, including memory and learning processes. In the present study, we have tested the hypothesis that GR brain targets are responsive to ultradian GC rhythmicity. We have used adrenalectomised rats replaced with pulses of corticosterone to determine the transcriptional effects of ultradian pulses in the hippocampus. Confocal microscopy confirmed that each GC pulse results in transient GR nuclear localisation in hippocampal CA1 neurones. Concomitant GR activation and DNA binding was demonstrated by synthetic glucocorticoid response element oligonucleotide binding, and verified for the Clock gene Period 1 promoter region by chromatin immunoprecipitation assays. Strikingly each GC pulse induced a 'burst' of transcription of Period 1 measured by heterogeneous nuclear RNA quantitative polymerase chain reaction. The net effect of pulsatile GC exposure on accumulation of the mature transcript was also assessed, revealing a plateau of mRNA levels throughout the time course of pulsatile exposure, indicating the pulse timing works optimally for steady state Per1 expression. The plateau dropped to baseline within 120 min of the final pulse, indicating a relatively short half-life for hippocampal Per1. The significance of this strict temporal control is that any perturbation to the pulse frequency or duration would have rapid quantitative effects on the levels of Per1. This in turn could affect hippocampal function, especially circadian related memory and learning processes.

Increase of fat oxidation and weight loss in obese mice caused by chronic treatment with human growth hormone or a modified C-terminal fragment.

OBJECTIVE: To observe the chronic effects of human growth hormone (hGH) and AOD9604 (a C-terminal fragment of hGH) on body weight, energy balance, and substrate oxidation rates in obese (ob/ob) and lean C57BL/6Jmice. In vitro assays were used to confirm whether the effects of AOD9604 are mediated through the hGH receptor, and if this peptide is capable of cell proliferation via the hGH receptor. METHOD: Obese and lean mice were treated with hGH, AOD or saline for 14 days using mini-osmotic pumps. Body weight, caloric intake, resting energy expenditure, fat oxidation, glucose oxidation, and plasma glucose, insulin and glycerol were measured before and after treatment. BaF-BO3 cells transfected with the hGH receptor were used to measure in vitro 125I-hGH receptor binding and cell proliferation. RESULTS: Both hGH and AOD significantly reduced body weight gain in obese mice. This was associated with increased in vivo fat oxidation and increased plasma glycerol levels (an index of lipolysis). Unlike hGH, however, AOD9604 did not induce hyperglycaemia or reduce insulin secretion. AOD9604 does not compete for the hGH receptor and nor does it induce cell proliferation, unlike hGH. CONCLUSIONS: Both hGH and its C-terminal fragment reduce body weight gain, increase fat oxidation, and stimulate lipolysis in obese mice, yet AOD9604 does not interact with the hGH receptor. Thus, the concept of hGH behaving as a pro-hormone is further confirmed. This data shows that fragments of hGH can act in a manner novel to traditional hGH-stimulated pathways.

Growth hormone (GH)-independent dimerization of GH receptor by a leucine zipper results in constitutive activation.

Growth hormone initiates signaling by inducing homodimerization of two GH receptors. Here, we have sought to determine whether constitutively active receptor can be created in the absence of the extracellular domain by substituting it with high affinity leucine zippers to create dimers of the growth hormone receptor (GHR) signaling domain. The entire extracellular domain of the GHR was replaced by the hemagglutinin-tagged zipper sequence of either the c-Fos or c-Jun transcription factor (termed Fos-GHR and Jun-GHR, respectively). Transient transfection of Fos-GHR or Jun-GHR resulted in activation of the serine protease inhibitor 2.1 promoter in Chinese hamster ovary-K1 cells to a level equal to that achieved by fully activated wild type GHR. Furthermore, stable expression of Jun-GHR alone or Fos-GHR and Jun-GHR together in the interleukin 3-dependent BaF-B03 cell line resulted in cell proliferation after interleukin 3 withdrawal at a rate equal to maximally stimulated wild type GHR-expressing cells. Activation of STAT 5b was also observed in Fos-Jun-GHR-expressing cells at a level equal to that in chronically GH-treated GHR-expressing cells. Thus, forced dimerization of the transmembrane and cytoplasmic domains of the GHR in the absence of the extracellular domain can lead to the constitutive activation of known GH signaling end points, supporting the view that proximity of Janus kinase 2 (JAK2) kinases is the essential element in signaling. Such constitutively active GH receptors may have particular utility for transgenic livestock applications.

Aspartate 171 is the major primate-specific determinant of human growth hormone. Engineering porcine growth hormone to activate the human receptor.

It has been known for more than 4 decades that only primate growth hormones are effective in primate species, but it is only with the availability of the 2.8 A structure of the human growth hormone (hGH).hGH-binding protein (hGHBP)2 complex that Souza and co-workers (Souza, S. C., Frick, G. P., Wang, X., Kopchick, J. J., Lobo, R. B., and Goodman, H. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 959-963) were able to provide evidence that Arg-43 on the primate receptor is responsible. Here we have examined systematically the interaction between Arg-43 (primate receptor) or Leu-43 (non-primate receptors) and their complementary hormone residues Asp-171 (primate GH) and His-170 (non-primate hormones) in a four-way comparison involving exchanges of histidine and aspartate and exchanges of arginine and leucine. BAF/B03 lines were created and characterized which stably expressed hGH receptor, R43L hGH receptor, rabbit GH receptor, and L43R rabbit GH receptor. These were examined for site 1 affinity, for the ability to bind intact cells, and for proliferative biopotency using hGH, D171H hGH, porcine GH, or H170D porcine GH. We find that the single interaction between Arg-43 and His-170/171 is sufficient to explain virtually all of the primate species specificity, and this is congruent with the crystal structure. Accordingly, for the first time we have been able to engineer a non-primate hormone to bind to and activate the human GH receptor.