Beta mRNA Levels Research Articles

Homocysteine Enhances Bone Resorption by Stimulation of Osteoclast Formation and Activity Through Increased Intracellular ROS Generation

Hyperhomocystinemia is a modifiable risk factor for osteoporosis and fracture. Physiologic concentrations of Hcy directly activate osteoclast formation and activity through stimulation of p38 MAPK and integrin beta3. The effects of Hcy were mediated by generation of intracellular ROS. Hyperhomocysteinemia is a modifiable risk factor for osteoporosis and its related bone fractures. It has been reported that bone resorption and turnover rate were increased in hyperhomocystinemia. Using mouse bone marrow cells, we examined the direct effects of homocysteine (Hcy) on osteoclast formation and activity. Osteoclast formation was determined by TRACP staining and TRACP activity. Intracellular reactive oxygen species (ROS) generation was measured using a fluorescent probe, dichlorodihydrofluorescein diacetate. Intracellular signaling cascades of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-kappaB were measured by Western blotting. Integrin beta3 mRNA levels were measured by RT-PCR. Actin ring formation and bone resorption assays were also performed. Physiologic concentrations of Hcy upregulated TRACP+ multinucleated cells and TRACP activity, stimulated actin ring formation, and increased the number of nuclei per cell and the level of expression of integrin beta3 mRNA. In addition, Hcy increased bone resorption and stimulated p38 MAPK activity and intracellular reactive oxygen species (ROS) generation. All of these Hcy-induced changes were blocked by pretreatment with the antioxidant, N-acetyl cysteine. Hcy directly activates osteoclast formation and activity through increased generation of intracellular ROS. These findings suggest that, in individuals with mild to moderate hyperhomocystinemia, increased bone resorption by osteoclasts may contribute to osteoporosis and that an antioxidant may attenuate bone loss in these individuals.

Evolution of renal function and Na+, K+-ATPase expression during ischaemia-reperfusion injury in rat kidney

The aim of the present work was to study the effects of an unilateral ischaemic-reperfusion injury on Na+, K+-ATPase activity, alpha1 and beta1 subunits protein and mRNA abundance and ATP content in cortical and medullary tissues from postischaemic and contralateral kidneys. Right renal artery was clamped for 40 min followed by 24 and 48 h of reperfusion. Postischaemic and contralateral renal function was studied cannulating the ureter of each kidney. Postischaemic kidneys after 24 (IR24) and 48 (IR48) hours of reperfusion presented a significant dysfunction. Na+, K+-ATPase alpha1 subunit abundance increased in IR24 and IR48 cortical tissue and beta1 subunit decreased in IR48. In IR24 medullary tissue, alpha1 abundance increased and returned to control values in IR48 while beta1 abundance was decreased in both periods. Forty minutes of ischaemia without reperfusion (I40) promoted an increment in alpha1 mRNA in cortex and medulla that normalised after 24 h of reperfusion. beta1 mRNA was decreased in IR24 medullas. No changes were observed in contralateral kidneys. This work provides evidences that after an ischaemic insult alpha1 and beta1 protein subunit abundance and mRNA levels are independently regulated. After ischaemic-reperfusion injury, cortical and medullary tissue showed a different pattern of response. Although ATP and Na+, K+-ATPase activity returned to control values, postischemic kidney showed an abnormal function after 48 h of reflow.

Neurogenic niche modulation by activated microglia: transforming growth factor β increases neurogenesis in the adult dentate gyrus

Adult neural stem cells (NSC) proliferate and differentiate depending on the composition of the cellular and molecular niche in which they are immersed. Until recently, microglial cells have been ignored as part of the neurogenic niche. We studied the dynamics of NSC proliferation and differentiation in the dentate gyrus of the hippocampus (DG) and characterized the changes of the neurogenic niche in adrenalectomized animals (ADX). At the cellular level, we found increased NSC proliferation and neurogenesis in the ADX animals. In addition, a morphologically distinct subpopulation of NSC (Nestin+/GFAP-) with increased proliferating profile was detected. Interestingly, the number of microglial cells at stages 2 and 3 of activation correlated with increased neurogenesis (r2 = 0.999) and the number of Nestin-positive cells (r2 = 0.96). At the molecular level, transforming growth factor beta (TGF-beta) mRNA levels were increased 10-fold in ADX animals. Interestingly, TGF-beta levels correlated with the amount of neurogenesis detected (r2 = 0.99) and the number of stage 2 and 3 microglial cells (r2 = 0.94). Furthermore, blockade of TGF-beta biological activity by administration of an anti-TGF-beta type II receptor antibody diminished the percentage of 5-bromo-2'-deoxyuridine (BrdU)/PSA-NCAM-positive cells in vivo. Moreover, TGF-beta was able to promote neurogenesis in NSC primary cultures. This work supports the idea that activated microglial cells are not pro- or anti-neurogenic per se, but the balance between pro- and anti-inflammatory secreted molecules influences the final effect of this activation. Importantly, we identified an anti-inflammatory cytokine, TGF-beta, with neurogenic potential in the adult brain.

Effects of estradiol on spatial learning, hippocampal cytochrome P450 19, and estrogen alpha and beta mRNA levels in ovariectomized female mice

The brain is an important target organ for peripherally synthesized estrogen but it also has its own steroid biosynthesis producing estrogen from testosterone catalyzed by the aromatase enzyme. This study examined the effects of estrogen treatment in two spatial memory tasks, one-arm-baited radial arm maze and a position discrimination task in the T-maze in ovariectomized female mice. Hippocampal cytochrome P450 19 (encoding aromatase), and estrogen receptor α and β gene expressions were also measured using real time quantitative polymerase chain reaction analysis. Estrogen (17β-estradiol) was administered either tonically via s.c. minipellets or phasically via daily i.p. injections. In ovariectomized mice, the tonic estrogen decreased the number of reference memory errors in radial arm maze. Tonic estrogen treatment also up-regulated the expression of cytochrome P450 19 and estrogen receptors. In contrast, estrogen injections decreased the expression of cytochrome P450 19 and estrogen receptor α genes. The number of reference memory errors correlated negatively with estrogen receptor α expression. These findings indicate that peripheral estrogen levels affect neuronal estrogen synthesis by regulating the cytochrome P450 19 gene expression and also influence estrogen receptor α expression. The results also suggest that tonic rather than cyclic estrogen treatment might be more beneficial for cognitive functions.

L-type calcium channel blocker suppresses calcineurin signal pathway and development of right ventricular hypertrophy.

Many studies have shown that L-type calcium channel blockers can prevent and treat right ventricular hypertrophy (RVH). In order to identify the mechanism, we investigated the role of the calcineurin signal pathway in the progression of RVH induced by chronic hypoxia and the effects of an L-type calcium channel blocker on the pathway. Rats were allocated to 1 of 3 groups (n=10 for each): chronic hypoxia group, amlodipine treatment group (30 mg/kg/day, administered via gavage); and control group. Rats in the amlodipine treatment group and the chronic hypoxia group were exposed to normobaric chronic hypoxia (9.5%-10.5% oxygen). We investigated the changes of right ventricle (RV) to left ventricle (LV) and interventricular septum (S) weight ratio [RV/(LV+S)], RV to body weight (BW) ratio (RV/BW), calcineurin A beta (CnAbeta) mRNA levels, cardiac myosin heavy chain beta (beta-MHC) mRNA levels and protein expression of CnAbeta, nuclear factor 3 of activated T cell (NFAT3), and beta-MHC. After 21 days, RV/(LV+S) and RV/BW were significantly higher in the chronic hypoxia group than in the control group and the amlodipine group (p<0.01). The expression of CnAbeta mRNA and protein, NFAT3 protein, beta-MHC mRNA and protein in RV of the chronic hypoxia group was higher than that of the control group and the amlodipine treatment group (p<0.01). The calcineurin signal pathway plays a critical role in the progression of RVH induced by chronic hypoxia. L-type calcium channel blockade suppresses the development of RVH by inhibiting this pathway.

Human Gingival Fibroblast Integrin Subunit Expression on Titanium Implant Surfaces

Implant surface characteristics have been shown to modify cell behavior and regulate integrin expression. Integrin expression and resultant integrin-mediated cellular activity are essential components of tissue healing and homeostasis. Although both osseous and soft tissue healing around dental implants are critical to clinical success, there is limited information available on the effect of implant surfaces on integrin expression in soft tissues. Therefore, the aim of this study was to examine integrin expression for gingival fibroblasts on titanium surfaces and the influence of titanium surface roughness on integrin expression and cell morphology. Human gingival fibroblasts were cultured on smooth (polished) and rough (sand-blasted acid-etched) titanium surfaces and a cell culture plastic (control) surface. To analyze integrin expression, total RNA was isolated from experimental and control cells, and levels of integrin subunit mRNA were assessed by reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for the alpha2, alpha4, alpha5, alpha(v), and beta1 integrin subunits and aldolase (internal control). PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE), confirmed via DNA sequencing, and quantified using computer-assisted densitometry. The expression of the integrin subunits was analyzed at the protein level using flow cytometry, as well as fluorescence and confocal laser microscopy. Cell morphology was evaluated using scanning electron microscopy (SEM). Our experiments demonstrated cellular expression of the alpha2, alpha4, alpha5, alpha(v), and beta1 integrin subunits at both mRNA and protein levels on all surfaces. In addition, the alpha4 and beta1 mRNA levels were significantly increased on smooth titanium relative to plastic surfaces (P <.05) with intermediate mRNA levels found on the rough titanium surfaces. The smooth titanium surfaces exhibited a flat monolayer of cells, while rough titanium surfaces showed cells orienting themselves along surface irregularities. These results demonstrate the presence of multiple integrin subunits in human gingival fibroblasts grown in contact with titanium implant surfaces and that titanium surface roughness alters cellular morphology but appears to have limited effects on integrin expression. This study provides insight into the complicated cellular and molecular events occurring at the implant surface that may be critical to optimizing the soft tissue interactions with the soft tissue-implant interface.

Connexin‐specific cell‐to‐cell transfer of short interfering RNA by gap junctions

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase beta (pol beta) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol beta was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mbeta16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mbeta16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol beta. These two pol beta knockdown cell lines (NRK-kcdc and Mbeta16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mbeta16tsA-wt cells or N2A cells. The levels of pol beta mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mbeta16tsA-kcdc cells with Mbeta16tsA-wt, N2A or NRK-wt cells had no effect on pol beta levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol beta levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol beta in the wt cells. The inability of Mbeta16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

Dexamethasone up‐regulates skeletal muscle maximal Na+,K+ pump activity by muscle group specific mechanisms in humans

Dexamethasone, a widely clinically used glucocorticoid, increases human skeletal muscle Na+,K+ pump content, but the effects on maximal Na+,K+ pump activity and subunit specific mRNA are unknown. Ten healthy male subjects ingested dexamethasone for 5 days and the effects on Na+,K+ pump content, maximal activity and subunit specific mRNA level (alpha1, alpha2, beta1, beta2, beta3) in deltoid and vastus lateralis muscle were investigated. Before treatment, maximal Na+,K+ pump activity, as well as alpha1, alpha2, beta1 and beta2 mRNA levels were higher (P < 0.05) in vastus lateralis than in deltoid. Dexamethasone treatment increased Na+,K+ pump maximal activity in vastus lateralis and deltoid by 14 +/- 7% (P < 0.05) and 18 +/- 6% (P < 0.05) as well as Na+,K+ pump content by 18 +/- 9% (P < 0.001) and 24 +/- 8% (P < 0.01), respectively. Treatment with dexamethasone resulted in a higher alpha1, alpha2, beta1 and beta2 mRNA expression in the deltoid (P < 0.05), but no effects on Na+,K+ pump mRNA were detected in vastus lateralis. In conclusion, dexamethasone treatment increased maximal Na+,K+ pump activity in both vastus lateralis and deltoid muscles. The relative importance of transcription and translation in the glucocorticoid-induced regulation of Na+,K+ pump expression seems to be muscle specific and possibly dependent on the actual training condition of the muscle, such that a high Na+,K+ pump maximal activity and mRNA level prior to treatment prevents the transcriptional response to dexamethasone, but not the increase in Na+,K+ pump content and maximal activity.

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.

Estrogen-related receptor in reproductive organs.

Estrogen-related receptor (ERR) was studied in the placenta and uterine endometrium, especially endometrial cancers, among reproductive organs. In the placenta, the estrogen receptor (ER) alpha and beta mRNA levels increased from the first to the second trimester, and then decreased until normal term delivery. Estrogen-related receptor alpha, beta and gamma mRNA levels gradually increased up to the second trimester, and then comparatively rapidly increased until normal term delivery. In endometrial cancers, ER alpha and beta mRNA levels decreased with clinical stage, myometrial invasion and dedifferentiation. Estrogen-related receptor alpha levels increased with clinical stage and myometrial invasion, and the ERR gamma levels increased with myometrial invasion. Estrogen-related receptors can bind to the steroid receptor coactivator family without any ligands, and drive transcription activity of the target genes. The manner of ERR and ER gene expressions might show a competitive interaction associated with the use of common cofactors. It is speculated that the upregulation of ERR is related to the placental growth after the downregulation of ER from the second trimester until delivery, and that ERR alpha and gamma are candidates for prognostic factors in endometrial cancer, although ERR are not directly related to tumor growth and advancement of endometrial cancer. (Reprod Med Biol 2005; 4: 129-131).

Pro-inflammatory Responses in Chicken Spleen and Brain Tissues after Infection with Very Virulent Plus Marek's Disease Virus

In chickens infected with virulent (v) or very virulent (vv) Marek's disease (MD) virus (MDV) strains, small to moderate increases in plasma nitric oxide (NO) levels are seen, respectively, whereas very virulent plus (vv+) strains induce very high levels in vivo. The data presented in this report show that chickens presenting with clinical neurological disease following infection with the vv+ RK-1 strain have significantly higher in vivo NO levels compared to RK-1-infected non-symptomatic chickens. Using quantitative real-time PCR (qPCR) assays, DNA was used to measure MDV copy numbers in the spleen and brain of P2a (MD-susceptible) and N2a (MD-resistant) chickens following infection with the JM-16 (v) or RK-1 (vv+) strains. RNA was used to measure inducible NO synthase (iNOS), interferon-gamma (IFN-gamma), interleukin (IL)-1beta, IL-6, and IL-8 mRNA levels, in addition to MDV-specific mRNA expression using quantitative RT-PCR (qRT-PCR) assays. Viral DNA loads were found to be considerably higher in RK-1-infected chickens than JM-16-infected chickens at most time points in both organs, with viral copy numbers being two to four logs lower in the brain. Large increases in iNOS, IFN-alpha, IL-1beta, IL-6, and IL-8 were seen in the brains of RK-1-infected chickens. These data strongly support the hypothesis that pro-inflammatory responses, including high levels of iNOS/NO, IFN-alpha, and pro-inflammatory cytokine expression in the chicken brain, may play a major role in the neurological diseases associated with vv+MDV strains.

Aberrant expression of transforming growth factor β‐1 (TGFβ‐1) per se does not discriminate fibrotic from non‐fibrotic chronic myeloproliferative disorders

Transforming growth factor beta-1 (TGF beta-1) is a potent inducer of fibrosis and has been shown to be essential for the development of bone marrow fibrosis in an animal model of idiopathic myelofibrosis (IMF). IMF belongs to the Philadelphia chromosome negative chronic myeloproliferative disorders (Ph(-) CMPD). Megakaryocytes and platelets have been suggested as the major cellular source of TGF beta-1 in IMF. The osteoclastogenesis inhibitory factor osteoprotegerin (OPG) seems to be regulated by TGF beta-1 and substantial involvement of OPG expression in the process of osteosclerosis in IMF has recently been suggested. In order to determine TGF beta-1 expression in IMF and other Ph(-) CMPD, total bone marrow cells as well as laser-microdissected megakaryocytes were quantitatively analysed by real-time RT-PCR. OPG mRNA expression in fibrotic IMF was correlated with TGF beta-1 mRNA expression in a case-specific manner. Both OPG and TGF beta-1 were detected immunohistochemically in order to delineate cellular origin. When total bone marrow cells were investigated, TGF beta-1 mRNA expression was increased in some but not all cases of IMF (n = 21), with highest values in fibrotic cases. Unexpectedly, increased values were also observed in essential thrombocythaemia (ET, n = 11) when compared to non-neoplastic haematopoiesis (n = 38). Megakaryocytes isolated by laser microdissection displayed elevated TGF beta-1 mRNA levels in most of the CMPD samples with no significant differences discernible between fibrotic IMF, polycythaemia vera (PV) and ET. TGF beta-1 protein was predominantly expressed by the myeloid lineage in Ph(-) CMPD and non-neoplastic haematopoiesis, which, however, displayed lower expression. IMF cases with advanced fibrosis concomitantly overexpressed TGF beta-1 and OPG. Immunohistochemically, OPG expression was found in different stromal cells and a subfraction of megakaryocytes. In conclusion, enhanced TGF beta-1 expression occurs in megakaryocytes as well as myeloid cells in Ph(-) CMPD. TGF beta-1 may be necessary, but is not sufficient, to induce bone marrow fibrosis in IMF because non-fibrotic Ph(-) CMPD entities share this feature with IMF and cannot be discriminated from each other on the basis of TGF beta-1 expression.

Estrogen-related receptor expression in placenta throughout gestation

Estrogen receptor (ER) alpha and beta mRNA levels increased from the first to the second trimester and then decreased until normal term delivery. Estrogen-related receptor (ERR) alpha, beta and gamma mRNA levels gradually increased up to the second trimester and then comparatively rapidly increased until normal term delivery. ERRs can bind to the steroid receptor coactivator family without any ligands and drive transcription activity of the target genes. The manner of ERR and ER gene expressions might show a competitive interaction associated with the use of common cofactors. It is speculated that the up-regulation of ERRs is related to placental growth after the down-regulation of ERs because of the remarkably high concentration of estrogens for ERs from the second trimester until delivery.

GnRH Antagonist-induced Down-regulation of the mRNA Expression of Pituitary Receptors: Comparisons with GnRH Agonist Effects

In order to compare the mechanism for the down regulation of the mRNA expression of pituitary receptors induced by GnRH antagonist (GnRHant) to that by GnRH agonist (GnRHa), we examined the effects of GnRHant (Cetrorelix, 333 mug/kg/day), GnRHa (leuprolide depot, 333 microg/kg), and GnRHant combined with GnRHa on LH response to exogenous GnRH, pituitary LH content, LH beta subunit mRNA, and GnRH receptor (GnRH-R) mRNA levels at 2, 5, 24, 72 hours, and 7 days after the treatment in ovariectomized rats. GnRHant significantly decreased serum LH, the LH response of the pituitary to exogenous GnRH, and the pituitary LH content compared to the control treatment, though GnRHa significantly increased serum LH. GnRHant with GnRHa significantly diminished the GnRHa-induced flare-up phenomenon. GnRHant significantly decreased LH beta mRNA and GnRH-R mRNA levels, but the magnitude of the decrease in these mRNA levels by GnRHant was significantly less than those by GnRHa until 72 hours following treatment. Prolonged treatment of GnRHant caused a marked inhibition of LH beta mRNA and GnRH-R mRNA expression, similar to that caused by GnRHa. Combination treatment with GnRHa and GnRHant was demonstrated to decrease LH beta mRNA and GnRH-R mRNA levels as much as GnRHa alone and GnRHant alone over 7 days of the treatment. The present study showed differences between GnRHant and GnRHa treatment in the reduction of GnRH-R mRNA levels up to 72 hours after the treatment, and indicated that the suppression of GnRH-R mRNA by GnRHant was the maximal by GnRHa 7 days after the treatment because more profound suppression was not observed upon additional treatment with GnRHa. The findings in the present study support the hypothesis that the mechanism by which GnRHant leads to down-regulation of the mRNA expression of pituitary receptors is similar to that of GnRHa.

Diesel exhaust affects immunological action in the placentas of mice

We investigated the effect of diesel exhaust (DE) on pregnancy and fetal development in mice at day 14 postcoitum (pc) with a special focus on the placenta. The number of absorbed fetuses increased in groups exposed to DE, and congestion was observed in histological sections of placentas. During placental absorption expression of CYP1A1 mRNA decreased to undetectable levels, whereas expression of TNF alpha mRNA increased approximately twofold over that of the control. Levels of CYP1A1 mRNA in normal placentas from DE-exposed mice were unchanged. mRNA levels of inflammatory cytokines IL-2, IL-5, IL-12 alpha, IL-12 beta, and GM-CSF increased in placentas exposed to DE (0.3 and 3.0 mg diesel exhaust particles (DEP)/m3). Expression of IL-5 mRNA was markedly increased in DE-exposed placentas, although levels were barely detectable in control placentas. IL-6 mRNA expression was increased approximately 10-fold in placentas exposed to DE (3.0 mg DEP/m3). It has been reported that expression of mRNA encoding proteins involved in immune function in the placenta is increased during fetal absorption in mice. In the present study, expression of such mRNA by the placenta was increased by DE exposure. Because it is believed that expression of mRNA in the placenta also affects fetal development, DE may promote fetal absorption. These findings suggest that in mice exposure to DE affects fetal absorption and placental function by modifying expression of immune-related genes during early gestation and expression of endocrine-related genes during late gestation.

Estrogen and progesterone regulate α, β, and γENaC subunit mRNA levels in female rat kidney

Estrogen and progesterone regulate alpha, beta, and gamma amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA levels in female rat kidney. Renal Na(+) handling differs between males and females. Further, within females Na(+) metabolism changes during the menstrual cycle and pregnancy. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated transport of Na(+) via the amiloride-sensitive Na(+) channel. This study examines the role of the female gender steroids in the regulation of expression of ENaC. We measured ENaC subunit mRNA levels in rat kidney using Northern blotting. Kidneys were taken from male and females at different ages and from adult ovariectomized rats treated with 17-beta-estradiol benzoate (estrogen) and/or progesterone for 8 or 24 hours. The abundance of alpha, beta, and gammaENaC mRNA was significantly higher in female compared to male rat kidneys from 10 weeks of age (P= 0.001, P= 0.004, and P= 0.02, N= 10, respectively). These differences were abolished in ovariectomized rats. Treatment of ovariectomized rats with estrogen increased alphaENaC mRNA abundance in the kidney at both 8 and 24 hours (P < 0.05, N= 6; and P < 0.05, N= 7, respectively). Progesterone inhibited the effect of estrogen on alphaENaC mRNA at 8 hours but when given alone increased gammaENaC mRNA (P < 0.05, N= 3). Neither hormone, alone or in combination, had any significant effect on betaENaC mRNA levels at 8 or 24 hours. Female gonadal steroids differentially modulate expression of ENaC subunit mRNA in the rat kidney.

Human thyroid carcinoma cell lines show different retinoic acid receptor repertoires and retinoid responses.

Disturbed expression of retinoic acid (RA) receptors (RAR/RXR) contributes to the pathogenesis and tumor progression of epithelial carcinomas. To examine whether altered responses to retinoids may correlate with differences in RA receptor equipment, retinoid effects were examined in human thyroid carcinoma cell lines of various differentiation stages in culture and after xenotransplantation onto rodent models. Cell growth was assessed by the MTT test, mRNA expression was examined by Northern blot and quantitative competitive RT-PCR, and type I 5'-deiodinase (5'DI) activity was measured by in vitro deiodination assay. Nude rats and mice were used for xenotransplantation experiments. All-trans-RA and RAR-selective synthetic retinoids stimulated activity and mRNA expression of the thyroid differentiation marker 5'DI in the follicular thyroid carcinoma cell line FTC-133. In the less differentiated FTC-238 cells, stimulation of 5'DI activity was less pronounced than in FTC-133 cells, and a reduced level of RAR beta mRNA was detected. In the anaplastic thyroid carcinoma cell lines HTh 74 and C 643, the activity of 5'DI was not increased by retinoids, and expression of RAR alpha mRNA was reduced. Proliferation of FTC-133 and FTC-238 cells was decreased by all-trans-RA. Pretreatment of FTC-133 with RA resulted in a reduced tumor growth in xenotransplantation experiments as compared with untreated control cells. This reduction was less pronounced in the case of FTC-238 cells. Thus, retinoid therapy might be applied to treat follicular thyroid carcinomas. However, tumor-specific RAR repertoires need to be analyzed as a prerequisite for successful intervention with appropriate, probably receptor-selective retinoids.

Modulation of neonatal immunological tolerance to ovalbumin by maternal essential fatty acid intake.

The present study examines whether dietary essential fatty acid (EFA) intake influences the induction of oral tolerance to ovalbumin (OA) in neonatal and adult rats. During late gestation and throughout lactation Sprague-Dawley rats were fed a diet supplemented (S) with EFA (7% soybean oil), or a diet deficient (D) in EFA (7% hydrogenated lard). The rat offspring were subsequently exposed to OA either via the milk at 10-16 days (neonatal rats), or as adults via the drinking water at 7-9 wk of age. Oral administration of OA to the adult rats lead to suppression of the delayed-type hypersensitivity (DTH) reactivity and IgG antibody response against OA, which was not influenced by their diets. In the offspring of the dams fed the D diet antigen exposure via the milk resulted in suppression of the serum antibody levels and DTH reaction against OA indicating induction of oral tolerance. Higher transforming growth factor beta (TGF-beta) mRNA levels in the draining lymph nodes suggested this to be mediated by regulatory T cells. In contrast, OA exposure of the dams fed the S diet did not result in a suppressed OA response of their offspring. Thus, the quality of FA ingested by the mother may have effects on the development of immunological tolerance to dietary antigens in the offspring. Our results might have importance for the understanding of the increase in allergy related to the Western type of diet.

Cloning of the cDNA for thyroid stimulating hormone beta subunit and changes in activity of the pituitary-thyroid axis during silvering of the Japanese eel, Anguilla japonica.

The purposes of this study were: (1) to clone the cDNA encoding pituitary thyroid-stimulating hormone beta subunit (TSH beta) of the Japanese eel, Anguilla japonica, together with its genomic DNA sequence, for phylogenetic analysis, and to study the regulation of the TSH beta gene expression in cultured pituitaries; and (2) to investigate the transcript levels of pituitary TSH beta mRNA and the serum thyroxine profiles at different stages of ovarian development before and during silvering in the wild female eels. The maturity of female eels was divided into four stages, juvenile, sub-adult, pre-silver, and silver, based on skin color and oocyte diameter. The genomic DNA of the TSH beta subunit contains two introns and three exons, and the TSH beta protein possesses a putative signal peptide of 20 amino acids and a mature peptide of 127 amino acids. The amino acid sequence identities of TSH beta mature peptide of Japanese eel compared with those of teleosts and other vertebrates are: European eel (98.4%), salmonids (60.6-61.3%), carps (52.0-56.7%), sturgeon (48.4%), and tetrapods (42.9-45.2%). In in vitro studies of the regulation of TSH beta mRNA it was found that thyrotropin-releasing hormone increased while thyroxine decreased its expression. RT-PCR and real-time quantitative PCR analysis showed that the transcript levels of TSH beta subunit increased during eel silvering. The serum thyroxine levels also increased in parallel with TSH beta mRNA expression during silvering, supporting the hypothesis that the hypothalamus-pituitary-thyroid axis is correlated to silvering in the wild female Japanese eels.

TGF-beta1mRNA levels in renal cortex and peripheral blood mononuclear cells of diabetic rats

To investigate the effect of hyperglycemia and pioglitazone on TGF-beta(1) gene expression in peripheral blood mononuclear cells (PBMC) and renal cortex, and the correlation of TGF-beta(1)mRNA levels between PBMC and renal cortex in STZ induced diabetic rats. Fifty-four Sprague-Dawley rats were randomly divided into three groups: 18 normal control rats (group C), 18 diabetic rats (group D) and 18 diabetic rats treated with pioglitazone (20 mg x kg(-1)x d(-1), group DP). Six rats from each group were sacrificed at 2, 4, 8 weeks. TGF-beta(1)mRNA levels of PBMC and renal cortex were examined by RT-PCR+Slit hybridization analysis. TGF-beta(1)mRNA level of renal cortex in group D was significantly higher than that in group C at each time point (P<0. 05); TGF-beta(1)mRNA level of PMBC in group D was slightly higher than that of group C at 4 weeks, and significantly higher at 8 weeks (P=0.01). There was positive correlation of TGF -beta(1)mRNA level between PBMC and renal cortex before (r=0.83, P=0.02) and after pioglitazone treatment at 8 weeks (r=0.82, P=0.03). TGF-beta(1)mRNA level of PBMC may reflect the change of TGF-beta(1) gene expression of renal cortex in diabetic rats.