Biomedical subjects

S P Tam

Publications and source records attributed to S P Tam.

At least 19 recordsLinked to original sources

PubMed2003

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma.

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

Breast Neoplasms↗

PubMed2002

Cytokine regulation of syndecan-1 and -2 gene expression in human periodontal fibroblasts and osteoblasts.

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta 1), and interleukin (IL-1 beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta 1, separately and in combination, in the prolonged presence of IL-1 beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta 1 and decreased levels in response to IL-1 beta. The effect of IL-1 beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta 1, separately or in combination, in the presence of IL-1 beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta 1 or IL-1 beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta 1 on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1 beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Alveolar Process↗

PubMed2002

Endothelin-1 stimulates human colonic myofibroblast contraction and migration.

BACKGROUND: Although the contractile, migratory, and proliferative responses of subepithelial myofibroblasts to injury have been postulated to be important events in intestinal wound healing, contractile force generation and migration by these cells has not been investigated previously, and the signals that regulate proliferation by these cells are poorly understood. AIMS: The primary aim of this study was to test the hypothesis that the inflammatory mediator endothelin-1 modulates contraction, migration, and proliferation of intestinal myofibroblasts. We also sought to examine the signal transduction pathways which might underlie these putative effects. METHODS: Contraction, migration, proliferation, cytosolic [Ca(2+)], and myosin phosphorylation were measured in human colonic subepithelial myofibroblasts in the absence and presence of endothelin receptor agonists and antagonists. RESULTS: Endothelin-1, but not interleukin 1 alpha, interleukin 6, interleukin 8, interleukin 10, or tumour necrosis factor alpha, induced a rapid and robust generation of contractile force, which was associated with an increase in cytosolic [Ca(2+)] and myosin phosphorylation. Inhibition of rho associated kinase reduced endothelin-1 stimulated myosin phosphorylation and contractile force development. Endothelin-1 stimulated migration with a dose-response relationship similar to that observed for contraction. Endothelin A and B receptors mediated contraction while migration was mediated predominantly through endothelin B receptors. Platelet derived growth factor and serum, but not endothelin-1, induced proliferation. CONCLUSIONS: Endothelin-1 stimulates colonic subepithelial myofibroblast contraction and migration via endothelin receptor mediated myosin phosphorylation. These results support an important role for subepithelial myofibroblasts in the injury response of the gut and consequently intestinal wound repair.

Amides↗

PubMed2002

A prostaglandin f(2alpha) analog induces suppressors of cytokine signaling-3 expression in the corpus luteum of the pregnant rat: a potential new mechanism in luteolysis.

PRL and placental lactogen (PL) play key roles in maintaining the rodent corpus luteum through pregnancy. Suppressors of cytokine signaling (SOCS) have been shown to decrease cell sensitivity to cytokines, including PRL, and so here we have addressed the issue of whether luteolysis induced by prostaglandin F(2alpha) (PGF(2alpha)) might up-regulate SOCS proteins to inhibit PRL signaling. In d 19 pregnant rats, cloprostenol, a PGF(2alpha) analog, rapidly induced transcripts for SOCS-3 and, to a lesser extent, SOCS-1. We also found increased SOCS-3 protein in the ovary by immunoblot and in the corpus luteum by immunohistochemistry. Increased SOCS-3 expression was preceded by an increase in STAT3 tyrosine phosphorylation 10 min after cloprostenol injection and was maintained for 4 h, as determined by gel shift and immunohistochemistry. Induction of SOCS-3 was accompanied by a sharp decrease in active STAT5, as determined by gel-shift assay and by loss of nuclear localized STAT5. Four hours after cloprostenol administration, the corpus luteum was refractory to stimulation of STAT5 by PRL administration, and this was not due to down-regulation of PRL receptor. Therefore, induction of SOCS-3 by PGF(2alpha) may be an important element in the initiation of luteolysis via rapid suppression of luteotropic support from PL.

Animals↗

PubMed2001

Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through rho-kinase-mediated alterations in the acto-myosin cytoskeleton.

Although migration of stellate cells during hepatic injury is essential for wound-healing and fibrosis of the liver, the extracellular and intracellular signals that regulate stellate cell migration are incompletely understood. In this study we tested the hypothesis that wound-induced migration of stellate cells is modulated by endothelin-1 (ET-1) through rho-kinase-mediated alterations in the acto-myosin cytoskeleton. To address this hypothesis, a method was established for direct visualization of wound-induced migration of culture-activated stellate cells with subcellular resolution. Migration in response to wounding was characterized by (1) plasma membrane ruffling and protrusion into the wound, (2) lamellipodia formation at the leading edge, (3) focal adhesion and stress fiber assembly, and (4) myosin reorganization. Exogenous ET-1 accelerated wound-induced migration of stellate cells, but did not alter wound-induced proliferation. Experiments using ET-1 antagonists in the absence of exogenous ET-1 showed that wound-induced migration was also stimulated by endogenous ET-1. Selective inhibition of rho-associated kinase decelerated migration in response to wounding. Moreover, inhibition of rho-associated kinase was distinguished by abrogation of focal adhesion formation, stress fiber assembly, and myosin reorganization. This study shows that rho-kinase-dependent alterations in the acto-myosin cytoskeleton contribute to wound-induced stellate cell migration, which is accelerated by both exogenous and endogenous ET-1. Consequently, these results provide important new evidence suggesting that, migration of stellate cells is modulated by paracrine and autocrine ET-1 stimulation via the action of rho-kinase on the acto-myosin cytoskeleton.

Actomyosin↗

PubMed2001

Tissue-specific induction of SOCS gene expression by PRL.

The mechanisms whereby tissue sensitivity to PRL is controlled are not well understood. Here we report that expression of mRNA and protein for members of the SOCS/CIS/JAB family of cytokine signaling inhibitors is increased by PRL administration in ovary and adrenal gland of the lactating rat deprived of circulating PRL and pups for 24 h but not in mammary gland. Moreover, suckling increases SOCS mRNA in the ovary but not in the mammary gland of pup-deprived rats. Deprivation of PRL and pups for 48 h allows the mammary gland to induce SOCS genes in response to PRL administration, and this is associated with a decrease in basal SOCS-3 mRNA and protein expression to the level seen in other tissues, suggesting that SOCS-3 induced refractoriness related to filling of the gland. In reporter assays, SOCS-1, SOCS-3, and CIS, but not SOCS-2, are able to inhibit transactivation of the STAT 5-responsive beta-lactoglobulin promoter in transient transfection assays. Moreover, suckling results in loss of ovarian and adrenal responsiveness to PRL administered 2 h after commencement of suckling, as determined by STAT 5 gel shift assay. Immunohistochemistry was used to localize the cellular sites of SOCS-3 and CIS protein expression in the ovary and adrenal gland. We propose that induced SOCS-1, SOCS-3, and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges in the corpus luteum and adrenal cortex during lactation, but after pup withdrawal, the mammary gland is rendered unresponsive to PRL by increased levels of SOCS-3.

Adrenal Glands↗

PubMed2001

Regulation of human apolipoprotein A-I gene expression by equine estrogens.

Estrogen replacement therapies, such as conjugated equine estrogen (CEE, Premarin), reduce the risk of coronary heart disease among postmenopausal women. In the present study, a HepG2 stable cell line (HepG2/S) that harbors a luciferase reporter gene cassette with the human apolipoprotein A-I (apoA-I) promoter region was used to examine the activity of CEE components in modulating human apoA-I promoter activity. A number of estrogens modulated apoA-I promoter activity, with equilenin (Eqn) being the most potent. Eqn produced a 3-fold increase in apoA-I promoter activity and a similar increase in apoA-I mRNA without affecting its degradation rate. Nuclear runoff assays indicated that the transcription rate of the apoA-I gene was increased 2.5-fold in Eqn-treated cells. When HepG2/S cells were exposed to Eqn, apoA-I protein secretion increased by 80%, whereas the level of secreted apoA-II remained unchanged. Transient transfection studies with human apoA-I promoter constructs derived from pGL3-luciferase reporter plasmid were used to identify the cis-acting element involved in Eqn-mediated induction. The results demonstrated that the apoA-I electrophile/antioxidant response element (EpRE/ARE) might be responsible for the increase in apoA-I promoter activity by Eqn. Cotransfection experiments using estrogen receptor (ERalpha and/or ERbeta) expression vectors have indicated that neither receptor can potentiate the Eqn-mediated induction of apoA-I promoter activity. In addition, mobility shift analysis using antibody against either ERalpha or ERbeta cannot detect the presence of these receptors in the DNA-protein complex. The data indicate that Eqn can induce the promoter activity of the human apoA-I gene, leading to an increase in apoA-I mRNA levels and apoA-I protein secretion through an ER-independent pathway involving apoA-I EpRE/ARE.

Animals↗

PubMed2000

Identification of the human cytochrome P450, P450RAI-2, which is predominantly expressed in the adult cerebellum and is responsible for all-trans-retinoic acid metabolism.

Retinoids, particularly all-trans-retinoic acid (RA), are potent regulators of cell differentiation, cell proliferation, and apoptosis. The role of all-trans-RA during development and in the maintenance of adult tissues has been well established. The control of all-trans-RA levels in cells and tissues is regulated by the balance between its biosynthesis and its catabolism to inactive metabolites. The cytochrome P450 enzyme P450RAI (herein renamed P450RAI-1) is partially responsible for this inactivation of all-trans-RA. In this report, we describe the identification, molecular cloning, and characterization of a second related enzyme, P450RAI-2, which is also involved in the specific inactivation of all-trans-RA. Transiently transfected P450RAI-2 can convert all-trans-RA to more polar metabolites including 4-oxo-, 4-OH-, and 18-OH-all-trans-RA. Competition experiments with other retinoids suggest that all-trans-RA is the preferred substrate. The high level of expression of P450RAI-2, particularly in the cerebellum and pons of human adult brain, suggests a unique role for this enzyme in the protection of specific tissues from exposure to retinoids.

Amino Acid Sequence↗

PubMed1999

Growth hormone as a cytokine.

1. The growth hormone (GH) receptor was the first of the class 1 cytokine receptors to be cloned. It shares a number of structural characteristics with other family members and common signalling mechanisms based on common usage of the Janus kinase 2 (JAK2). 2. Growth hormone receptor activation is initiated by GH-induced homodimerization of receptor molecules. This has enabled the creation of specific hormone antagonists that block receptor dimerization. 3. The details of the transcription factors used by the activated receptor are being revealed as a result of promoter analyses and electrophoretic mobility gelshift analysis. 4. Growth hormone receptors are widespread and their discovery in certain tissues has led to the assignment of new physiological roles for GH. Some of these involve local or paracrine roles for GH, as befits its cytokine status. 5. Four examples of such novel roles are discussed. These are: (i) the brain GH axis; (ii) GH and the vitamin B12 axis; (iii) GH in early pre-implantation development; and (iv) GH in development of the tooth. 6. We propose that the view that GH acts through the intermediacy of insulin-like growth factor-1 is simplistic; rather, GH acts to induce an array of growth factors and their receptors and the composition of this array varies with tissue type and, probably, stage of development.

Animals↗

PubMed1999

Cardiac-specific overexpression of RhoA results in sinus and atrioventricular nodal dysfunction and contractile failure.

RhoA is a low-molecular-weight GTPase that has been implicated in the regulation of hypertrophic cardiac muscle cell growth. To study the role of RhoA in control of cardiac function in vivo, transgenic mice expressing wild-type and constitutively activated forms of RhoA under the control of the cardiac-specific alpha-myosin heavy chain promoter were generated. Transgene-positive mice expressing high levels of either wild-type or activated RhoA showed pronounced atrial enlargement and manifested a lethal phenotype, often preceded by generalized edema, with most animals dying over the course of a few weeks. Echocardiographic analysis of visibly healthy wild-type RhoA transgenic mice revealed no significant change in left ventricular function. As their condition deteriorated, significant dilation of the left ventricular chamber and associated decreases in left ventricular contractility were detected. Heart rate was grossly depressed in both wild-type and activated RhoA-expressing mice, even prior to the onset of ventricular failure. Electrocardiography showed evidence of atrial fibrillation and atrioventricular block. Interestingly, muscarinic receptor blockade with atropine did not elicit a positive chronotropic response in the transgenic mice. We suggest that RhoA regulates cardiac sinus and atrioventricular nodal function and that its overexpression results in bradycardia and development of ventricular failure.

Animals↗

PubMed1998

Human S mu binding protein-2 binds to the drug response element and transactivates the human apoA-I promoter: role of gemfibrozil.

Previously, we demonstrated that protein-DNA interactions at the drug response element (DRE) in the human apoA-I promoter were important for the induction of apoA-I gene expression by gemfibrozil. We now report the cloning and characterization of a DRE transactivating factor. The cloned protein is identical to the putative helicase and potential transcription factor human S mu binding protein-2 (HSmuBP2). It is also related to glial factor-1 (GF1), an incomplete version of HSmuBP2 lacking the first 494 and the last 128 amino acids. Gel mobility shift assays demonstrated that HSmuBP2 binds apoA-I DRE oligomers and forms a specific protein-DNA complex. Northern blot analysis showed that HSmuBP2 mRNA is expressed at various levels in a wide range of human tissues. Transient cotransfection experiments performed in HepG2 cells demonstrated that overexpression of HSmuBP2 or GF1 induced apoA-I proximal promoter activity by 3-fold and that the apoA-I DRE was necessary for transactivation. Additionally, we demonstrated that transactivation was increased a further 2- to 3-fold by exposing the cells to gemfibrozil. Together these observations indicate that HSmuBP2 acts as a transcription factor that regulates apoA-I gene expression in hepatoma cells and whose activity may be stimulated by gemfibrozil treatment.

Apolipoprotein A-I↗

PubMed1997

Regulation of human apolipoprotein A-I gene expression by gramoxone.

To induce oxidative stress, HepG2 cells were exposed to a compound known as gramoxone. This compound undergoes a one-electron reduction to form a stable free radical which is capable of generating reactive oxygen species. We demonstrated that exposure of HepG2 cells to gramoxone (0.1 microM) resulted in a 2-fold decrease in apoA-I mRNA with no significant change in apoB and apoE mRNA levels. To examine if increased rates of mRNA degradation were responsible for the reduction in apoA-I mRNA levels, mRNA half-lives were measured in the presence of actinomycin D with and without gramoxone treatment. These studies revealed a 4-fold increase in the rate of apoA-I mRNA degradation in cells exposed to gramoxone. In similarly treated cells, nuclear run-off assays indicated that the transcription rate of the apoA-I gene was also increased 2-fold. Consistent with nuclear run-off assays, transient transfection experiments using a series of pGL2-derived luciferase reporter plasmids containing the human apoAI proximal promoter demonstrated that gramoxone treatment increased apoA-I promoter activity 2-fold. We have identified a potential "antioxidant response element" (ARE) in the apoA-I promoter region that may be responsible for the increase in apoA-I transcriptional activity by gramoxone. Gel mobility shift assays with an ARE oligonucleotide revealed increased levels of a specific protein-DNA complex that formed with nuclear extracts from gramoxone-treated cells. UV cross-linking experiments with the ARE and nuclear extracts from either untreated or gramoxone-treated cells detected proteins of approximately 100 and 115 kDa. When a single copy of the ARE was inserted upstream of the SV40 promoter in a luciferase reporter plasmid, a significant 2-fold induction in luciferase activity was observed in HepG2 cells in the presence of gramoxone. In contrast, a plasmid containing a mutant apoAI-ARE did not confer responsiveness to gramoxone. Furthermore, pGL2 (apoAI-250 mutant ARE), in which point mutations eliminated the ARE in the apoAI promoter, showed no increase in luciferase activity in response to gramoxone. Taken together, the data suggest that gramoxone affects apoA-I mRNA levels by both transcriptional and post-transcriptional mechanisms.

Apolipoprotein A-I↗

PubMed1997

Effects of dimethyl sulfoxide on apolipoprotein A-I in the human hepatoma cell line, HepG2.

Exposure of HepG2 cells to 1% (v/v) dimethyl sulfoxide (DMSO), an effective free radical scavenger, for 24 h resulted in a 2-fold increase in the levels of apolipoprotein (apo) A-I mRNA and secreted protein, with no significant change in apoA-II, apoB, and apoE mRNA and protein levels. The induction of apoA-I was accompanied by a 50% increase in secreted HDL. Nuclear run-off assays indicated that the transcription rate of the apoA-I gene was also increased 2-fold in DMSO-treated cells. Consistent with nuclear run-off assays, transient transfection experiments, using a series of pGL2-derived luciferase reporter constructs containing the human apoA-I proximal promoter, demonstrated that DMSO treatment increased apoA-I promoter activity 2-fold. We have identified a potential 'antioxidant response element' (ARE) in the apoA-I promoter that may be responsible for the increase in apoA-I transcriptional activity by DMSO. Gel mobility shift assays with an apoA-I-ARE revealed increased levels of a specific protein-DNA complex that formed with nuclear extracts from DMSO-treated cells. The formation of this complex is sequence specific as determined by DNA competition studies. When a copy of the ARE was inserted upstream of the SV40 promoter in a luciferase reporter plasmid, a significant 2-fold induction in luciferase activity was observed in HepG2 cells in the presence of DMSO. In contrast, a plasmid containing a mutated apoA-I-ARE did not confer responsiveness to DMSO treatment. Furthermore, pGL2 (apoA-I-250 mutant ARE), in which point mutations eliminated the ARE in the apoA-I promoter, showed no increase in luciferase activity in response to DMSO. These results implicate protein-DNA interactions at the antioxidant response element region in the transcriptional induction of human apoA-I gene expression by DMSO.

Antioxidants↗

PubMed1996

Protein-DNA interactions at a drug-responsive element of the human apolipoprotein A-I gene.

Previously, we demonstrated that when two human hepatoma cell lines, Hep3B and HepG2, were exposed to gemfibrozil, a hypolipidemic drug, a 2-fold induction in apolipoprotein A-I (apoA-I) mRNA levels resulted. To determine if mRNA stabilization was responsible for the changes in apoA-I mRNA levels, the half-lives for apoA-I mRNA were measured in the presence of actinomycin D with and without gemfibrozil. These experiments revealed no differences in stability. However, nuclear run-off assays indicated that the transcription rate of the apoA-I gene was increased 2-fold in gemfibrozil-treated cells. Transient transfection experiments also indicated that the induction of apoA-I mRNA level in response to gemfibrozil is mediated at the transcriptional level. We have identified two copies of the "drug-responsive element" (DRE) in the apoA-I promoter region that may be responsible for the increase in apoA-I transcriptional activity by gemfibrozil. Using gel mobility shift assays with a synthetic DRE oligonucleotide, we have demonstrated that exposure of Hep3B and HepG2 cells to gemfibrozil resulted in strong induction of a protein-DNA complex. The formation of this complex is highly sequence-specific as indicated by the DNA competition experiments. The drug-inducible nuclear proteins bind to the DRE of the human apoA-I gene with an apparent Kd of 4.1 nM. Methylation interference experiments have localized the contact sites of nuclear factors to the DRE region. Southwestern blot analyses have identified two groups of drug-inducible nuclear proteins with molecular masses of approximately 30 and 15 kDa. When a copy of synthetic DRE oligonucleotide was inserted upstream of the thymidine kinase promoter and luciferase reporter construct, a significant 2-fold induction in luciferase activity was observed in the presence of gemfibrozil following transient transfection of two human hepatoma cell lines, HepG2 and Hep3B. However, a plasmid containing one copy of mutated apoA-I-DRE oligomer did not confer responsiveness to gemfibrozil treatment. Furthermore, pGL2 (apoA-I -250 mutant DRE), which carried an internal mutation of the DRE in the human apoA-I proximal promoter region, showed no increase in luciferase activity in response to gemfibrozil. These results implicate protein-DNA interactions at the DRE region in the transcriptional induction of human apoA-I gene expression by gemfibrozil.

Apolipoprotein A-I↗

PubMed1996

Gene expression of the receptor for growth-hormone-releasing hormone is physiologically regulated by glucocorticoids and estrogen.

We investigated the effects of glucocorticoids and estrogen on the gene expression of growth hormone (GH) and the receptor for growth-hormone-releasing hormone (GHRH) by measuring the mRNA levels of GH and GHRH receptor in pituitary tissues of Sprague-Dawley rats using Northern blot hybridization and specific cDNA probes. Male rats, 6 weeks of age, were either adrenalectomized (or sham-operated) or treated with varying doses of dexamethasone (40, 200, 500 or 1,000 micrograms/kg/day, i.p.) for 3 days. Female rats, 4 weeks of age, were oophorectomized or sham-operated, and treated with 17 beta-estradiol benzoate 25 micrograms/kg/day (or vehicle) s.c. for 5 days starting 10 days after oophorectomy. Adrenalectomy was associated with a reduction in weight gain and decreased GHRH receptor mRNA levels (p < 0.05 and p < 0.0001 versus sham-operated, respectively). Dexamethasone treatment, however, was associated with a dose-dependent reduction in weight gain (p < 0.0001) but dose-dependent increases in GHRH receptor mRNA and GH mRNA levels (p < 0.0001 and p < 0.05, respectively). In the female rats, weight gain was increased by oophorectomy (p < 0.005 vs. sham-operated) and decreased by estrogen treatment (p < 0.05 vs. vehicle-treated). Pituitary GHRH receptor mRNA levels were also increased by oophorectomy (p < 0.05) and decreased by estrogen (p < 0.005). GH mRNA levels were unchanged by oophorectomy but decreased after estrogen treatment (p < 0.05). In conclusion, our findings suggest that endogenous glucocorticoids and estrogen are physiological regulators of pituitary GHRH receptor gene expression. Glucocorticoids and estrogen also regulate GH secretion via effects on GH gene expression. Changes in GHRH receptor and GH mRNA levels cannot explain the growth retardation in dexamethasone-treated rats.

Animals↗

PubMed1996

Gene expression of hypothalamic somatostatin, growth hormone releasing factor, and their pituitary receptors in hypothyroidism.

Thyroid hormones are important to growth in mammals and have been shown to rapidly stimulate the rate of GH gene transcription. In this study, we investigated whether thyroid hormones modulate GH secretion through their effects on the gene expression of GRF, somatostatin (SS), GRF receptor, and receptor subtype 2 for SS (SSTR2). Male adult Sprague-Dawley rats were rendered hypothyroid with a single injection of propylthiouracil followed by methimazole in drinking water (0.02%) for 1 day to 12 weeks. Total RNA extracted from the anterior pituitary and hypothalamus was analyzed by Northern hybridization. GH messenger RNA (mRNA) level in the anterior pituitary was significantly reduced in the hypothyroid animals (P < 0.0001 vs. controls for all treatment duration > or = 1 week). An increase in hypothalamic GRF mRNA level, by 2- and 4-fold, respectively, was seen after 3 and 12 weeks of antithyroid treatment (both P < 0.001 vs. controls). Hypothalamic GRF content, studied in 12-week hypothyroid rats only, was decreased compared with controls (P < 0.05). A reduction in pituitary GRF receptor mRNA level was observed after 1 week of antithyroid treatment (P < 0.01 after 1 week, P < 0.001 after 3 weeks). Total hypothalamic SS content and SS mRNA level in hypothalamic fragments consisting predominantly of the paraventricular and periventricular nuclei became significantly decreased (P < 0.05 and P < 0.005 respectively) after 12-weeks of antithyroid treatment. The reduction in SS gene expression in the periventricular nuclei was confirmed by in situ hybridization. No significant change in the mRNA level of pituitary SSTR2 was observed up to 12 weeks of antithyroid treatment. In conclusion, we have demonstrated a reduction in the gene expression of GRF receptor and SS in the hypothyroid rat. Our results suggest that the changes in hypothalamic GRF and SS gene expression in hypothyroid rats may be compensatory in nature and are likely to be secondary to the reduction in GH synthesis and secretion in these animals. The reduction in basal and GRF-stimulated GH secretion in hypothyroidism can be explained by the observed reduction in GH and GRF receptor gene expression.

Animals↗

PubMed1996

Interaction of a recombinant form of apolipoprotein[a] with human fibroblasts and with the human hepatoma cell line HepG2.

We have studied the binding, uptake, and degradation of a recombinant form of apolipoprotein[a] (r-apo[a]) using a cultured cell model. In HepG2 cells and in human fibroblasts, r-apo[a] complexed with low density lipoprotein(LDL) is bound and internalized via high affinity (Kd = 10 nM) receptors; in both cell types, low affinity (Kd = 200-300 nM) sites also mediate free apo[a] uptake. Using competition studies, we found that the high affinity binding component corresponds to the LDL receptor. Involvement of the LDL receptor in r-apo[a] uptake by fibroblasts was confirmed using fibroblasts derived from an individual homozygous for familial hypercholesterolemia; in contrast to normal fibroblasts, these cells lacked the high affinity r-apo[a] binding component. Cell association of 125I-labeled r-apo[a] was increased and decreased concomitantly with the up- and down-regulation of the LDL receptor in response to a number of compounds. The addition of alpha 2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease total specific binding of r-apo[a], suggesting that neither the low density lipoprotein receptor-related protein nor cell surface proteoglycans are involved in r-apo[a] clearance. The low affinity binding component present in both fibroblasts and HepG2 cells likely corresponds to the plasminogen receptor, as binding of r-apo[a] to these sites was specifically decreased by the addition of plasminogen or the lysine analogue epsilon-aminocaproic acid, but not by the addition of tissue-type plasminogen activator. Heparin abolished uptake of r-apo[a] by the LDL receptor component only; this indicates that apo[a] must be associated with LDL to be cleared by this receptor. In contrast, free apo[a] can be effectively cleared by the plasminogen receptor which may represent a significant route of clearance for free apo[a] in vivo.

Aminocaproic Acid↗

PubMed1994

Regulation of apolipoprotein A-I gene expression by phenobarbital in the human hepatocarcinoma cell line, Hep3B.

Apolipoprotein (apo) A-I is the major protein constituent of plasma high density lipoprotein (HDL), which has been suggested to play a protective role against the development of atherosclerosis. The effect of phenobarbital on apo A-I mRNA and protein levels was studied in the human hepatoma cell line, Hep3B. Exposure of Hep3B cells to the drug (200 micrograms/ml) for 16 h resulted in a 4-fold and 8-fold increase in apo A-I mRNA and secreted protein levels, respectively. The induction of apo A-I mRNA level caused by phenobarbital could be due to increased rates of transcription and/or alteration in mRNA stability. To test these possibilities, nuclear run-off transcription assays and pulse-chase deinduction experiments were performed. We have demonstrated that phenobarbital treatment is associated with a 2-fold induction in apo A-I transcriptional activity. The estimated half-lives for apo A-I mRNA are 2 h and 3.6 h in the absence or presence of phenobarbital, respectively. The combination of increase in apo A-I transcription rate and mRNA stabilization could explain the 4-fold induction in apo A-I mRNA levels caused by phenobarbital treatment. However, these events could not be solely responsible for the 8-fold increase in secreted apo A-I protein level observed. The results suggest that the mechanism(s) by which phenobarbital induces apo A-I production operate at both pre- and either co- or post-translational mechanisms. The induction of apo A-I is specific since no significant alteration in apo E mRNA and proteins was observed in drug-treated cells.

Animals↗