Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.
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- Abstract: Atherosclerosis x
- Abstract: Heart x
- Abstract: Liver x
- Abstract: Hypertensive x
- Abstract: Vasculature x
- Abstract: Angiotensin x
- Abstract: Inflammation x
- Abstract: Mineralocorticoid x
- Abstract: Cardiac* x
- Abstract: myocardial x
- Abstract: Cardio* x
MK O'Bryan, KL Sebire, O Gerdprasert, MP Hedger, MT Hearn and DM de Kretser
PS Leung and PO Carlsson
The classical concept of the renin-angiotensin system (RAS) is that of a blood-borne cascade, whose final and bioactive product, angiotensin II, plays an important endocrine role in the maintenance of blood pressure and electrolyte as well as fluid balance. In addition to this circulating RAS, there are an increasing number of studies to suggest the existence of a local angiotensin-generating system in several tissues. The so-called tissue RAS can act locally as a paracrine and/or autocrine factor in meeting specific needs for individual tissues and it can operate, in whole or in part, independently of the circulating counterpart. Recent studies on the expression and localization of key RAS components, particularly angiotensinogen and renin, have provided solid evidence for the existence of an intrinsic, angiotensin-generating system in the pancreas. The tissue RAS has a potential role in finely regulating exocrine and endocrine functions of the pancreas such as ductal anion secretion and islet hormonal secretion. Some of these effects may be exerted via the markedly vasoconstrictive effects of RAS. Of particular interest in this context are the recent epidemiological data showing that administration of angiotensin-converting enzyme inhibitors appears to be protective against the development of diabetes in hypertensive patients. Moreover, the upregulation of pancreatic RAS has been shown to occur during chronic hypoxia. The significance of changes in pancreatic RAS could have a potential role in acute pancreatitis, islet transplantation and in different shock states, by causing a further decrease of blood perfusion in the pancreas.
The nuclear receptor family responds to a diverse group of ligands, including steroids, retinoids, thyroid hormone, prostaglandins and fatty acids. Previous sequence analyses of adrenal and sex steroid receptors indicate that they form a clade separate from other nuclear receptors. However, the relationships of adrenal and sex steroid receptors to each other and to their ancestors are not fully understood. We have used new information from androgen, estrogen, mineralocorticoid and progesterone receptors in fish to better resolve the phylogeny of adrenal and sex steroid receptors. Sequence divergence between fish and mammalian steroid receptors correlates with differences in steroid specificity, suggesting that phylogeny needs to be considered in evaluating the endocrine effects of xenobiotics. Among the vertebrate steroid receptors, the most ancient is the estrogen receptor. The phylogeny indicates that adrenal and sex steroid receptors arose in a jawless fish or a protochordate and that changes in the sequence of the hormone-binding domain have slowed considerably in land vertebrates. The retinoid X receptor clade is closest to the adrenal and sex steroid receptor clade. Retinoid X receptor is noteworthy for its ability to form dimers with other nuclear receptors, an important mechanism for regulating the action of retinoid X receptor and its dimerization partners. In contrast, the adrenal and sex steroid receptors bind to DNA as homodimers. Moreover, unliganded adrenal and sex steroid receptors form complexes with heat shock protein 90. Thus, the evolution of adrenal and sex steroid receptors involved changes in protein-protein interactions as well as ligand recognition.
FM Rogerson, YZ Yao, BJ Smith, N Dimopoulos and PJ Fuller
Spironolactone is a mineralocorticoid receptor (MR) antagonist in clinical use. The compound has a very low affinity for the glucocorticoid receptor (GR). Determinants of binding specificity of spironolactone to the MR were investigated using chimeras created between the ligand-binding domains (LBDs) of the MR and the GR. These chimeras had previously been used to investigate aldosterone binding specificity to the MR. Spironolactone was able to compete strongly for [(3)H]-aldosterone and [(3)H]-dexamethasone binding to a chimera containing amino acids 804-874 of the MR, and weakly for [(3)H]-dexamethasone binding to a chimera containing amino acids 672-803 of the MR. Amino acids 804-874 were also critical for aldosterone binding specificity. Models of the MR LBD bound to aldosterone and spironolactone were created based on the crystal structure of the progesterone receptor LBD. The ligand-binding pocket of the MR LBD model consisted of 23 amino acids and was predominantly hydrophobic in nature. Analysis of this model in light of the experimental data suggested that spironolactone binding specificity is not governed by amino acids in the ligand-binding pocket.
Shirlene X Ong, Keefe Chng, Michael J Meaney and Jan P Buschdorf
During pregnancy, glucocorticoids transfer environmental signals to the growing brain and its associated neuroendocrine system to modulate their maturation and function during adolescence and adulthood. Increased in utero exposure to glucocorticoids is associated with impaired fetal growth resulting in low birth weight (LBW) and compromised neural development. The underlying molecular changes affecting brain development, however, are largely unknown. Here, we compared the relative mRNA expression of genes directly involved in glucocorticoid signaling in the hippocampus, amygdala, and cortex of female non-human primate neonates (Macaca fascicularis) of naturally occurring normal birth weight and LBW. We focused on the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) genes as well as that for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and found a significantly decreased MR:GR mRNA ratio in the hippocampus and lower expression of 11β-HSD1 in the amygdala associated with LBW. The MR:GR mRNA ratio in the amygdala and cortex was not associated with birth weight, reflecting tissue-specific effects. Protein quantification in the hippocampus confirmed our finding of a decreased hippocampal MR:GR ratio. Our data suggest that the MR:GR ratio in the hippocampus and the expression of 11β-HSD1 in the amygdala are associated with intrauterine growth restriction in non-human primates during early perinatal development.
M M Pasquarette, P M Stewart, M L Ricketts, K Imaishi and J I Mason
The type 2 isoform of 11 β-hydroxysteroid dehydrogenase (11β-HSD2), which catalyzes the conversion of cortisol to hormonally inactive cortisone in man, is principally expressed in the placenta and mineralocorticoid target tissues, kidney and colon. To date, few studies have addressed the regulation of this novel 11β-HSD2 isoform. We have characterized the nature and regulation of the 11β-HSD activity expressed in a human cytotrophoblastic cell line, the JEG-3 choriocarcinoma cell. The 11β-HSD activity in JEG-3 cell homogenates required NAD+ as cofactor with NADP ineffective and demonstrated a high affinity for cortisol (apparent K m 31 nm). Incubation of JEG-3 cells with forskolin and dibutyryl cyclic AMP increased 11β-HSD2 activity several-fold in a time-dependent manner, while treatment with phorbol ester had little, if any, effect on 11β-HSD2 activity. Northern blot analysis of RNA isolated from JEG-3 cells after these treatments demonstrated a marked increase in a 1·9 kb 11β-HSD2 mRNA species in cells treated with forskolin for 24 h. We conclude that 11β-HSD2 is regulated by activation of the protein kinase A pathway, but not the protein kinase C pathway in human choriocarcinoma cells, and that this regulation occurs at a pretranslational level. JEG-3 cells provide an excellent model for further studies on the regulation of 11β-HSD2 gene expression in human trophoblast tissue.
M R Thomas, J P Miell, A M Taylor, R J M Ross, J R Arnao, D E Jewitt and A M McGregor
Thyroid hormones are essential for the normal growth and development of many tissues. In the rat, hypothyroidism is associated with growth impairment, and hyperthyroidism with the development of a hypercatabolic state and skeletal muscle wasting but, paradoxically, cardiac hypertrophy. The mechanism by which thyroid hormone produces cardiac hypertrophy and myosin isoenzyme changes remains unclear. The role of IGF-I, an anabolic hormone with both paracrine and endocrine actions, in producing cardiac hypertrophy was investigated during this study in hyperthyroid, hypothyroid and control rats. A treated hypothyroid group was also included in order to assess the effect of acute normalization of thyroid function.
Body weight was significantly lower in the hyperthyroid (mean±s.e.m.; 535·5±24·9 g, P<0·05), hypothyroid (245·3±9·8 g, P<0·001) and treated hypothyroid (265·3±9·8 g, P<0·001) animals when compared with controls (618·5±28·6 g). Heart weight/body weight ratios were, however, significantly increased in the hyperthyroid (2·74 ± 0·11×10−3, P<0·01) and treated hypothyroid (2·87±0·07 ×10−3, P<0·001) animals when compared with controls (2·26±0·03 × 10−3). Serum IGF-I concentrations were similar in the control and hyperthyroid rats (0·91±0·07 vs 0·78±0·04 U/ml, P=0·26), but bioactivity was reduced by 70% in hyperthyroid serum, suggesting a circulating inhibitor of IGF. Serum IGF-I levels (0·12±0·03 U/ml, P<0·001) and bioactivity (0·12±0·04 U/ml, P<0·001) were significantly lower in the hypothyroid group. Liver IGF-I mRNA levels were not statistically different in the control and hyperthyroid animals, but were significantly reduced in the hypothyroid animals (P<0·05 vs control). Heart IGF-I mRNA levels were similar in the control and hypothyroid rats, but were significantly increased in the hyperthyroid and treated hypothyroid animals (increased by 32% in hyperthyroidism, P<0·05; increased by 57% in treated hypothyroidism, P<0·01). Cardiac IGF-I was significantly elevated in hyperthyroidism (0·16±0·01 U/mg heart tissue, P<0·01), was low in hypothyroidism (0·08±0·01 U/mg, P<0·01) and was normalized in the treated hypothyroid group (0·11 ± 0·01 U/mg vs control, 0·13±0·01 U/mg).
Low body mass during both hypothyroidism and hyperthyroidism is therefore associated with reduced systemic IGF bioactivity. In hypothyroidism there is a primary defect in the endocrine function of IGF-I, while in hyperthyroidism serum IGF bioactivity is reduced in the presence of normal endocrine production of this anabolic hormone. In contrast, the paracrine actions of IGF-I are increased in the heart during hyperthyroidism, and this hormone appears to play a part in the development of hyperthyroid cardiac hypertrophy.
Tae Woo Jung, Yoon Hee Chung, Hyoung-Chun Kim, A M Abd El-Aty and Ji Hoon Jeong
Leukocyte cell-derived chemotaxin 2 (LECT2) is a recently identified novel hepatokine that causes insulin resistance in skeletal muscle by activating c-Jun N-terminal kinase (JNK), thereby driving atherosclerotic inflammation. However, the role of LECT2 in inflammation and insulin resistance in adipocytes has not been investigated. In this study, we report that LECT2 treatment of differentiated 3T3-L1 cells stimulates P38 phosphorylation in a dose-dependent manner. LECT2 also enhanced inflammation markers such as IκB phosphorylation, nuclear factor kappa beta (NF-κB) phosphorylation and IL-6 expression. Moreover, LECT2 treatment impaired insulin signaling in differentiated 3T3-L1 cells, as evidenced by the decreased levels of insulin receptor substrate (IRS-1) and Akt phosphorylation and reduced insulin-stimulated glucose uptake. Furthermore, LECT2 augmented lipid accumulation during 3T3-L1 cell differentiation by activating SREBP1c-mediated signaling. All these effects were significantly abrogated by siRNA-mediated silencing of P38, CD209 expression or a JNK inhibitor. Our findings suggest that LECT2 stimulates inflammation and insulin resistance in adipocytes via activation of a CD209/P38-dependent pathway. Thus, these results suggest effective therapeutic targets for treating inflammation-mediated insulin resistance.
Pia Kiilerich, Gérard Triqueneaux, Nynne Meyn Christensen, Vincent Trayer, Xavier Terrien, Marc Lombès and Patrick Prunet
The salmonid corticosteroid receptors (CRs), glucocorticoid receptors 1 and 2 (GR1 and GR2) and the mineralocorticoid receptor (MR) share a high degree of homology with regard to structure, ligand- and DNA response element-binding, and cellular co-localization. Typically, these nuclear hormone receptors homodimerize to confer transcriptional activation of target genes, but a few studies using mammalian receptors suggest some degree of heterodimerization. We observed that the trout MR confers a several fold lower transcriptional activity compared to the trout GRs. This made us question the functional relevance of the MR when this receptor is located in the same cells as the GRs and activated by cortisol. A series of co-transfection experiments using different glucocorticoid response elements (GREs) containing promoter-reporter constructs were carried out to investigate any possible interaction between the piscine CRs. Co-transfection of the GRs with the MR significantly reduced the total transcriptional activity even at low MR levels, suggesting interaction between these receptors. Co-transfection of GR1 or GR2 with the MR did not affect the subcellular localization of the GRs, and the MR-mediated inhibition seemed to be independent of specific activation or inhibition of the MR. Site-directed mutagenesis of the DNA-binding domain and dimerization interface of the MR showed that the inhibition was dependent on DNA binding but not necessarily on dimerization ability. Thus, we suggest that the interaction between MR and the GRs may regulate the cortisol response in cell types where the receptors co-localize and propose a dominant-negative role for the MR in cortisol-mediated transcriptional activity.
P A Weller, M C Dickson, N S Huskisson, M J Dauncey, P J Buttery and R S Gilmour
Genomic DNA encoding the 5′ region of the porcine IGF-I gene was cloned and sequenced and shown to be highly homologous to that of man, rats and sheep. Two leader exons (exons 1 and 2), which are alternately spliced to exon 3 (encoding part of the mature IGF-I molecule), were identified by RNase protection analysis. In both cases, transcription initiates upstream from exons 1 and 2 at multiple dispersed start sites to yield two distinct IGF-I mRNA transcript classes (1 and 2) which differ in the precursor peptides predicted from their individual leader sequences.
The expression of class 1 and 2 transcripts was measured in liver and muscle RNA from two groups of 2-month-old pigs whose energy status had been manipulated within physiological limits to produce marked differences in plasma IGF-I levels and growth rates. For this purpose, RNase protection probes were developed that contained the individual leader exons 1 and 2 linked separately to the common exon 3, so that class-specific and total IGF-I gene expression could be determined in a single assay. At normal plasma IGF-I concentrations (200 ng/ml), class 1 and 2 transcripts comprised 81 and 19% respectively of total liver IGF-I mRNA, while at a lower plasma concentration (90 ng/ml) the corresponding values were 95 and 5% respectively. Although both classes of transcript declined with the decrease in plasma IGF-I, the relative drop in levels of class 2 transcripts (84%) was substantially greater than that of class 1 (54%). In longissimus dorsi, cardiac and soleus muscles IGF-I mRNA was predominantly of class 1 and did not change in response to decreased plasma IGF-I. This suggests that liver-derived endocrine IGF-I has an important function in the regulation of muscle growth and that class 2 IGF-I transcripts are more sensitive to conditions that promote optimal growth.