Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor

Glucocorticoids are widely used in the treatment of inflammatory and other diseases. However, high-dose or chronic administration often triggers troublesome side effects such as metabolic syndrome and osteoporosis. We recently described that one glucocorticoid receptor gene produces eight translational glucocorticoid receptor isoforms that have distinct gene-regulatory abilities. We show here that specific, but not all, glucocorticoid receptor isoforms induced apoptosis in human osteosarcoma U-2 OS bone cells. Whole human genome microarray analysis revealed that the majority of the glucocorticoid target genes were selectively regulated by specific glucocorticoid receptor isoforms. Real-time PCR experiments confirmed that proapoptotic enzymes necessary for cell death, granzyme A and caspase-6, were induced by specific glucocorticoid receptor isoforms. Chromatin immunoprecipitation assays further suggested that glucocorticoid receptor isoform-dependent induction of proapoptotic genes was likely due to selective coregulator recruitment and chromatin modification. Interestingly, the capabilities to transrepress proinflammatory genes were similar among glucocorticoid receptor isoforms. Together, these findings provide new evidence that translational glucocorticoid receptor isoforms can elicit distinct glucocorticoid responses and may be useful for the development of safe glucocorticoids with reduced side effects.     

Transcriptional regulation by the nuclear receptor superfamily

In the past year, additional experimental data have expanded our understanding of the molecular mechanisms that underlie nuclear receptor control of regulatory programs. It is increasingly clear -that steroid members (e.g. glucocorticoid and estrogen) and non-steroid members (e.g. retinoic acid, thyroid hormone, and vitamin D) of the nuclear receptor superfamily may utilize distinct strategies in achieving their complex control of gene regulation.     

Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing.

We here report the existence of 6 additional isoforms of the NMDA receptor generated via alternative splicing by molecular analysis of cDNA clones isolated from a rat forebrain cDNA library. These isoforms possess the structures with an insertion at the extracellular amino-terminal region or deletions at two different extracellular carboxyl-terminal regions, or those formed by combinations of the above insertion and deletions. One of the deletions results in the generation of a new carboxyl-terminal sequence. All these isoforms possess the ability to induce electrophysiological responses to NMDA and respond to various antagonists selective to the NMDA receptor in the Xenopus oocyte expression system. In addition, a truncated form of the NMDA receptor also exists that contains only the extreme amino-terminal sequence of this protein molecule. These data indicate that the NMDA receptor consists of heterogeneous molecules that differ in the extracellular sequence of the amino- and carboxyl-terminal regions.     

Negative regulation of growth hormone receptor signaling

GH has been of significant scientific interest for decades because of its capacity to dramatically change physiological growth parameters. Furthermore, GH interacts with a range of other hormonal pathways and is an established pharmacological agent for which novel therapeutical applications can be foreseen. It is easy to see the requirement for a number of postreceptor mechanisms to regulate and control target tissue sensitivity to this versatile hormone. In recent years, some of the components that take part in the down-regulatory mechanism targeting the activated GH receptor (GHR) have been defined, and the physiological significance of some of these key components has begun to be characterized. Down-regulation of the GHR is achieved through a complex mechanism that involves rapid ubiquitin-dependent endocytosis of the receptor, the action of tyrosine phosphatases, and the degradation by the proteasome. The suppressors of cytokine signaling (SOCS) protein family, particularly SOCS2, plays an important role in regulating GH actions. The aim of this review is to summarize collected knowledge, including very recent findings, regarding the intracellular mechanisms responsible for the GHR signaling down-regulation. Insights into these mechanisms can be of relevance to several aspects of GH research. It can help to understand growth-related disease conditions, to explain GH resistance, and may be used to develop pharmaceuticals that enhance some the beneficial actions of endogenously secreted GH in a tissue-specific manner.     

Biological activities of two porcine growth hormone-releasing hormone receptor isoforms

Binding of growth hormone-releasing hormone (GHRH) to two isoforms (G3R and G5R) of the porcine GHRH receptor was studied. Both G3R- and G5R-cDNA were isolated from a porcine anterior pituitary cDNA library and have an identical primary structure from aa 1 to 418 and a different aa sequence from aa 419 to 423. In addition, the G5R isoform contains an extra C-terminal tail of 28 aa. The G3R and G5R mRNAs arise from alternative splicing of a single precursor mRNA for GHRH receptors. A mammalian cell expression vector containing either G3R or G5R cDNA under the regulation of a strong human cytomegalovirus promoter was constructed and used to transfect a human embryonic kidney 293 cell line. Two stable transfectants (293/G3R-4 and 293/G5R-12) were isolated on the basis of high expression of the receptor mRNAs. Both G3R and G5R mRNAs were expressed at similarly high levels in 293/G3R-4 and 293/G5R-12 cells; however, GHRH binding to 293/G3R-4 cells was much greater than that observed for 293/G5R-12 cells. Basal as well as GHRH-stimulated GTPase activity and intracellular cAMP concentration are also significantly greater in 293/G3R-4 cells as compared to 293/G5R-12 cells. We conclude that the modification of GHRH receptor at the C-terminal region hindered GHRH binding to the receptor and thus attenuates its biological activities.     

Regulatory effects of growth hormone, glucocorticoids, and thyroid hormone on the estrogen receptor level in the rat liver.

The multihormonal regulation of the estrogen receptor in the liver of female rats was studied under in vivo conditions. The steroid receptor level was assayed by hormone binding and specific mRNA analyzed by solution hybridization using a 35S-labeled RNA probe complementary to the ligand-binding domain of the estrogen receptor gene. Serum growth hormone levels were measured and correlated to the effects of glucocorticoid and thyroid hormone administration on the estrogen receptor expression. In animals subjected to adrenalectomy plus thyroidectomy, the estrogen receptor concentration was reduced from 59 fmol/mg cytosol protein to 10 fmol/mg protein (i.e., with 87% relative to control animals). Adrenalectomy or thyroidectomy alone caused a decrease with 14% and 66%, respectively. Substitution with 10 micrograms betamethasone and 1 microgram triiodothyronine daily for 9 days completely restored the receptor content to control levels. Substitution with either hormone alone increased, but only partially restored receptor levels. The effect of betamethasone alone was dose dependent from 10 micrograms/d to 100 micrograms/d. This dose dependence was not seen when the animal simultaneously received 1 microgram of triiodothyronine. Superphysiologic doses of triiodothyronine did not raise estrogen receptor levels above those seen in animals treated with physiologic doses. High doses of triiodothyronine (greater than 20 micrograms/d) decreased serum growth hormone levels. The estrogen receptor mRNA levels in livers from hypophysectomized animals were increased after treatment with growth hormone (2.5-fold), thyroid hormone (two-fold), and glucocorticoids (1.5-fold). The results obtained indicate a very complex regulation of liver estrogen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The expression of two isoforms of the human fibroblast growth factor receptor (flg) is directed by alternative splicing.

Structural analysis of the cDNA for the human fibroblast growth factor receptor (flg) revealed the existence of a larger and a shorter isoform of the receptor. The larger form has three extracellular immunoglobulin-like domains. On the other hand, the shorter form deletes the first (the most external) immunoglobulin-like domain region. Two consecutive amino acids (Arg Met) between the first and second immunoglobulin-like domains are sometimes deleted from the shorter form. In this paper, we isolated and analyzed the gene for the human fibroblast growth factor receptor. Organization of the gene revealed that the isoforms are produced by two different types of alternative splicing (the cassette and internal donor types) from the common gene. In human placenta, the shorter form is expressed as the major isoform.