Amrita Kamat

Mechanisms in tissue-specific regulation of estrogen biosynthesis in humans.

In humans, aromatase P450, which catalyses conversion of C(19)-steroids to estrogens, is expressed in several tissues, including gonads, brain, adipose tissue, skin and placenta, and is encoded by a single-copy gene (CYP19); however, this does not hold true for all species. The human gene is approximately 130 kb and its expression is regulated, in part, by tissue-specific promoters and by alternative splicing mechanisms. Using transgenic mouse technology, it was observed that ovary-, adipose tissue- and placenta-specific expression of human CYP19 is directed by relatively small segments of DNA within 500 bp upstream of each of the tissue-specific first exons. Thus, the use of alternative promoters allows greater versatility in tissue-specific regulation of CYP19 expression. Characterization and identification of transcription factors and crucial cis-acting elements within genomic regions that direct tissue-specific expression will contribute to improved understanding of the regulation of CYP19 expression in the tissues that synthesize estrogens under both physiological and pathophysiological conditions.

Estrogen receptor α (ERα) mediates stimulatory effects of estrogen on aromatase (CYP19) gene expression in human placenta

A 246-bp region upstream of placenta-specific exon I.1 of the human aromatase (hCYP19) gene mediates placenta-specific, developmental, and O(2) regulation of expression. In this study, trophoblast differentiation and associated induction of CYP19 expression were prevented when cytotrophoblasts were cultured in phenol red-free medium containing charcoal-stripped serum or with the estrogen receptor (ER) antagonist, ICI 182,780, suggesting a stimulatory role of estrogen/ER. ERalpha protein was expressed in human trophoblasts and increased during syncytiotrophoblast differentiation, whereas ERbeta was undetectable. Mutational analysis revealed that an estrogen response element-like sequence (ERE-LS) at -208 bp is required for inductive effects of estradiol/ERalpha on hCYP19I.1 promoter activity in transfected COS-7 cells. Increased binding of syncytiotrophoblast compared with cytotrophoblast nuclear proteins to the ERE-LS was observed in vitro; however, ERalpha antibodies failed to supershift the complex and in vitro-transcribed/translated ERalpha did not bind. Nonetheless, chromatin immunoprecipitation assays in cultured trophoblasts revealed recruitment of endogenous ERalpha to the -255- to -155-bp region containing the ERE-LS before induction of hCYP19 expression; this was inhibited by ICI 182,780. Chromatin immunoprecipitation also revealed increased acetylated histone H3(K9/14) and decreased methylated histone H3(K9) associated with this region during trophoblast differentiation. These modifications were prevented when trophoblasts were incubated with ICI 182,780, suggesting that ERalpha recruitment to the -255- to -155-bp region promotes histone modifications leading to increased hCYP19 transcription. Thus, during trophoblast differentiation, estrogen/ERalpha exerts a positive feedback role, which promotes permissive histone modifications that are associated with induction of hCYP19 gene transcription.

Blockade by lnterleukin-1-Alpha of Nitricoxidergic Control of Luteinizing Hormone-Releasing Hormone Release in vivo and in vitro

Nitric oxide (NO) synthase (NOS), the enzyme that converts arginine into citrulline plus NO, the latter a highly active free radical, occurs in a large number of neurons in the brain, including certain neurons in the hypothalamus. Our previous experiments have shown that norepinephrine (NE)-induced prostaglandin E2 (PGE2) release from medial basal hypothalamic explants (MBH) is mediated by NO. Because release of luteinizing hormone (LH)-releasing hormone (LHRH) is also driven by NE and PGE2, we hypothesized that NO controls pulsatile release of LHRH in vivo, which in turn induces pulsatile LH release. Indeed, in vivo and in vitro experiments using an inhibitor of NOS (NG-monomethyl-L-arginine; NMMA) demonstrated that pulsatile LH release is mediated by NO; LHRH release in vitro is also mediated by this free radical. Cytokines that are released from cells of the immune system during infection also inhibit LHRH release. We compared the action of one such cytokine, interleukin-1 alpha (IL-1 alpha), on LHRH release with that of substances which inhibit or induce NO release. Microinjection of IL-1 alpha (0.06 pmol in 2 microliters) into the third cerebral ventricle (3V) of conscious, castrated male rats had an action similar to that of 3V microinjection of NMMA (1 mg in 5 microliters): it blocked pulsatile LH, but not follicle-stimulating hormone (FSH) release. The only difference between the responses to NMMA and IL-1 alpha was that the latency to onset was greater with IL-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B.

Mesangioproliferative glomerulonephritis is associated with overactive PDGF receptor signal transduction. We show that the phytoalexin resveratrol dose dependently inhibits PDGF‐induced DNA synthesis in mesangial cells with an IC50 of 10 µM without inducing apoptosis. Remarkably, the increased SIRT1 deacetylase activity induced by resveratrol was not necessary for this inhibitory effect. Resveratrol significantly blocked PDGF‐stimulated c‐Src and Akt kinase activation, resulting in reduced cyclin D1 expression and attenuated pRb phosphorylation and cyclin‐dependent kinase‐2 (CDK2) activity. Furthermore, resveratrol inhibited PDGFR phosphorylation at the PI 3 kinase and Grb‐2 binding sites tyrosine‐751 and tyrosine‐716, respectively. This deficiency in PDGFR phosphorylation resulted in significant inhibition of PI 3 kinase and Erk1/2 MAPK activity. Interestingly, resveratrol increased the activity of protein tyrosine phosphatase PTP1B, which dephosphorylates PDGF‐stimulated phosphorylation at tyrosine‐751 and tyrosine‐716 on PDGFR with concomitant reduction in Akt and Erk1/2 kinase activity. PTP1B significantly inhibited PDGF‐induced DNA synthesis without inducing apoptosis. These results for the first time provide evidence that the stilbene resveratrol targets PTP1B to inhibit PDGFR mitogenic signaling.—Venkatesan, B., Ghosh‐Choudhury, N., Das, F., Mahimainathan, L., Kamat, A., Kasinath, B. S., Abboud, H. E., Choudhury, G. G. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B. FASEB J. 22, 3469–3482 (2008)

Glutamic acid induces luteinizing hormone releasing hormone release via alpha receptors

Glutamic acid (GA) and norepinephrine (NE) stimulate luteinizing hormone-releasing hormone (LHRH) release via release of nitric oxide (NO) from NOergic neurons in the arcuate-median eminence region. To determine if GA releases LHRH via direct stimulation of NOergic neurons, or via stimulation of noradrenergic terminals, arcuate median eminence explants from male rats were incubated with various compounds, and the LHRH release into the medium was measured. GA-induced release of LHRH was completely blocked by phentolamine (1 microM), an alpha receptor blocker, which, by itself, had no effect on the release. Nitroprusside (NP), which spontaneously releases NO, more than doubled LHRH release. To determine if alpha receptors on the LHRH neuron are required for the action of NP, the tissue was incubated with phentolamine, plus NP. Phentolamine had no effect on the LHRH-releasing action of NP. The results are interpreted to mean that GA activates the release of NE from the noradrenergic terminals. This acts on alpha receptors on the NOergic neuron to produce the release of NO. This NO diffuses to the LHRH terminals and induces release of LHRH.

Role of β-adrenergic receptors in regulation of hepatic fat accumulation during aging

Excessive fat accumulation in liver (hepatic steatosis) predisposes to hepatic functional and structural impairment and overall metabolic risk. Previous studies noted an association between hepatic steatosis and age in humans and rodents. However, the mechanisms leading to age-associated hepatic fat accumulation remain unknown. Earlier work from our group showed that β-adrenergic receptor (β-AR) levels and β-AR-stimulated adenylyl cyclase activity increase in rat liver during aging. Here we investigated whether age-associated increases in β-AR signaling play a role in augmenting hepatic lipid accumulation. We demonstrate an increase in hepatic lipid content during senescence and a significant correlation between hepatic fat content and stimulation of adenylyl cyclase activity by the β-AR agonist isoproterenol in rat liver. Isoproterenol administration to young and old rodents in vivo increased hepatic lipid accumulation. Furthermore, in vitro overexpression of β1- and β2-AR subtypes in hepatocytes from young rodents increased cellular lipid content, whereas inhibition of β-ARs by receptor subtype-specific inhibitors reduced lipid levels in hepatocytes from senescent animals. Isoproterenol-induced hepatic lipid accumulation in vivo was prevented by the β-AR nonselective blocker propranolol, suggesting a novel therapeutic effect of this class of drugs in hepatic steatosis. Acipimox, which inhibits adipose tissue lipolysis, did not alter isoproterenol-mediated hepatic fat accumulation; thus β-AR responsive hepatic lipid accumulation does not appear to be related primarily to altered lipolysis. These findings suggest that augmented hepatic β-AR signaling during aging may increase lipid accumulation in liver and advocate a possible role for β-adrenergic blockers in preventing or retarding the development of hepatic steatosis.

Impaired mitochondrial complex III and melatonin responsive reactive oxygen species generation in kidney mitochondria of db/db mice.

Abstract:  We have previously demonstrated that melatonin, at pharmacological concentrations, causes rapid reactive oxygen species (ROS) generation at the antimycin‐A sensitive site of mitochondrial complex III (MC‐3). In the current work, we used this melatonin response to investigate the role of mitochondrial dysfunction in the development of diabetic nephropathy. We find that the development of diabetic nephropathy, as indicated by hyperfiltration and histopathological lesions in the kidney of db/db mice, is associated with diminished melatonin‐induced ROS generation and MC‐3 activity, indicating impaired MC‐3 at the antimycin‐A site. The MC‐3 protein level in the renal mitochondria was equivalent in db/db and the nondiabetic db/m mice, whereas mitochondrial complex I (MC‐1) protein was dramatically upregulated in the db/db mice. This differential regulation in mitochondrial complexes may alter the equilibrium of the electron transport in renal mitochondria and contribute to ROS overproduction. The study provides one mechanism of enhanced oxidative stress that may be involved in the pathogenesis of diabetic nephropathy in db/db mice.

Liver mTOR Controls IGF-I Bioavailability by Regulation of Protein Kinase CK2 and IGFBP-1 Phosphorylation in Fetal Growth Restriction

Fetal growth restriction (FGR) increases the risk for perinatal complications and predisposes the infant to diabetes and cardiovascular disease later in life. No treatment for FGR is available, and the underlying pathophysiology remains poorly understood. Increased IGFBP-1 phosphorylation has been implicated as an important mechanism by which fetal growth is reduced. However, to what extent circulating IGFBP-1 is phosphorylated in FGR is unknown, and the molecular mechanisms linking FGR to IGFBP-1 phosphorylation have not been established. We used umbilical cord plasma of appropriate for gestational age (AGA) and growth-restricted human fetuses and determined IGFBP-1 and IGF-I concentrations (ELISA) and site-specific IGFBP-1 phosphorylation (Western blotting using IGFBP-1 phospho-site specific antibodies). In addition, we used a baboon model of FGR produced by 30% maternal nutrient restriction and determined mammalian target of rapamycin (mTOR)C1 activity, CK2 expression/activity, IGFBP-1 expression and phosphorylation, and IGF-I levels in baboon fetal liver by Western blot, enzymatic assay, and ELISA. HepG2 cells and primary fetal baboon hepatocytes were used to explore mechanistic links between mTORC1 signaling and IGFBP-1 phosphorylation. IGFBP-1 was hyperphosphorylated at Ser101, Ser119, and Ser169 in umbilical plasma of human FGR fetuses. IGFBP-1 was also hyperphosphorylated at Ser101, Ser119, and Ser169 in the liver of growth-restricted baboon fetus. mTOR signaling was markedly inhibited, whereas expression and activity of CK2 was increased in growth-restricted baboon fetal liver in vivo. Using HepG2 cells and primary fetal baboon hepatocytes, we established a mechanistic link between mTOR inhibition, CK2 activation, IGFBP-1 hyperphosphorylation, and decreased IGF-I-induced IGF-I receptor autophosphorylation. We provide clear evidence for IGFBP-1 hyperphosphorylation in FGR and identified an mTOR and CK2-mediated mechanism for regulation of IGF-I bioavailability. Our findings are consistent with the model that inhibition of mTOR in the fetal liver, resulting in increased CK2 activity and IGFBP-1 hyperphosphorylation, constitutes a novel mechanistic link between nutrient deprivation and restricted fetal growth.

Geldanamycin Derivative Ameliorates High Fat Diet-Induced Renal Failure in Diabetes

Diabetic nephropathy is a serious complication of longstanding diabetes and its pathogenesis remains unclear. Oxidative stress may play a critical role in the pathogenesis and progression of diabetic nephropathy. Our previous studies have demonstrated that polyunsaturated fatty acids (PUFA) induce peroxynitrite generation in primary human kidney mesangial cells and heat shock protein 90β1 (hsp90β1) is indispensable for the PUFA action. Here we investigated the effects of high fat diet (HFD) on kidney function and structure of db/db mice, a widely used rodent model of type 2 diabetes. Our results indicated that HFD dramatically increased the 24 h-urine output and worsened albuminuria in db/db mice. Discontinuation of HFD reversed the exacerbated albuminuria but not the increased urine output. Prolonged HFD feeding resulted in early death of db/db mice, which was associated with oliguria and anuria. Treatment with the geldanamycin derivative, 17-(dimethylaminoehtylamino)-17-demethoxygeldanamycin (17-DMAG), an hsp90 inhibitor, preserved kidney function, and ameliorated glomerular and tubular damage by HFD. 17-DMAG also significantly extended survival of the animals and protected them from the high mortality associated with renal failure. The benefit effect of 17-DMAG on renal function and structure was associated with a decreased level of kidney nitrotyrosine and a diminished kidney mitochondrial Ca2+ efflux in HFD-fed db/db mice. These results suggest that hsp90β1 is a potential target for the treatment of nephropathy and renal failure in diabetes.

Cellular signals underlying β-adrenergic receptor mediated salivary gland enlargement

We examined the cellular signaling pathways involved in parotid gland enlargement induced by repeated isoproterenol administration in rats. Immunoblot analysis revealed early (1h) activation of the mitogen activated protein kinase (MAPK) ERK1/2, and progressive activation of epidermal growth factor receptor (EGFR), p38MAPK and p70S6 kinase (p70S6K) during 72h of isoproterenol treatment. Expression of β-adrenergic receptors (ARs) of the β2, but not β1, subtype increased over time in parallel with increases in the proliferation marker PCNA and parotid gland weight. Levels of β2-AR mRNA, assessed by quantitative RT-PCR and Northern blot analysis, were upregulated in parotid glands of isoproterenol treated rats. cAMP response element binding protein (CREB), a positive regulator of β2-AR transcription, was activated at 1h after isoproterenol administration, as evidenced by increased nuclear translocation and DNA binding using immunohistochemical staining and electrophoretic mobility shift assay. ELISA of NF-κB, also a β2-AR transcriptional regulator, revealed an increase in p65 and p50 subunits in nuclear protein extracts from parotid glands of isoproterenol treated rats. Together, these results demonstrate that β-adrenergic stimulation activates diverse cell survival and progrowth signaling pathways, including cAMP and EGFR linked activation of ERK1/2, p38MAPK, and p70S6K, and also induction of β2-ARs, possibly mediated by CREB and NF-κB, resulting in salivary gland enlargement. We propose that during isoproterenol treatment activation of the β1-AR, the predominant β-AR subtype in unstimulated salivary glands, initiates proliferative signaling cascades, and that upregulation of the β2-AR plays an essential role in later stages of salivary gland growth.