Glutamatergic control of the expression of the proenkephalin gene in rat frontoparietal cortical slice cultures

The expression of the proenkephalin (PEnk) gene in rat neocortex develops during the first two postnatal weeks in an outside first- inside last mode that is opposite to the gradient of neurogenesis. To test whether the distribution of PEnk gene expression depends on the formation of the local circuitry, we examined the role of glutamate neurons in the expression of the gene in slice cultures of rat frontoparietal cortex. In situ and Northern blot hybridization were used for analysis. In slices explanted at postnatal day 6, the neuronal expression of the PEnk gene developed as in vivo. The expression responded to glutamate receptor agonists and antagonists in a time- dependent manner. After 2 days in vitro the expression of the gene was only enhanced by N-methyl-D-aspartate receptors, whereas after 7 days in vitro AMPA receptors also regulated the expression. We concluded that glutamate neurons are involved in the development and maintenance of the PEnk gene expression in the neocortex.      

Screening and identification of substances that regulate nephrin gene expression using engineered reporter podocytes

Downregulation of nephrin in podocytes leads to development of proteinuria in human and experimental kidney diseases. However, little is understood about pathophysiologic substances that regulate nephrin expression. In this report, we established conditionally immortalized reporter podocytes REPON for sensitive, continuous monitoring of nephrin gene expression. A murine podocyte cell line harboring a temperature-sensitive simian virus 40 large T antigen was stably transfected with a gene encoding secreted alkaline phosphatase (SEAP) under the control of the 5.4 or 8.3 kb nephrin gene promoter. The established reporter cells REPON5.4 and REPON8.3 were exposed to various pathophysiologic substances, and culture media were subjected to SEAP assay to identify regulators of nephrin gene expression. Among the bioactive substances tested, three physiological ligands of nuclear receptors including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and dexamethasone significantly activated the nephrin gene promoter in a dose-dependent manner. These effects were observed in both REPON5.4 and REPON8.3 and were associated with upregulation of nephrin mRNA. The effects of these substances were synergistic, and the maximum effect was observed by combination of three agents. In contrast, inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha as well as phorbol ester significantly downregulated the activity of the nephrin promoter as well as nephrin gene expression. These results elucidated the bidirectional regulation of nephrin by distinct pathophysiologic substances and may provide molecular bases for explaining how proteinuria is induced under pathologic situations and why some ligands for nuclear receptors have the anti-proteinuric potential.     

Glucose and insulin chronically regulate insulin action via different mechanisms in BC3H1 myocytes. Effects on glucose transporter gene expression.

We previously reported that, in primary cultured adipocytes, chronic exposure to glucose plus insulin impairs the insulin-responsive glucose transport system. In this study, we examined regulation of glucose transport in BC3H1 myocytes as a model for muscle and found important differences between BC3H1 cells and adipocytes. In myocytes, chronic glucose exposure per se (25 mM) decreased basal glucose transport activity by 78% and insulin's acute ability to maximally stimulate transport by 68% (ED50 approximately 2.5 mM; T1/2 approximately 4 h). D-Mannose and 3-O-methyl-glucose diminished transport rates with approximately 100 and 50% of the potency of D-glucose, respectively, whereas L-glucose, D-fructose, and D-galactose were inactive. Chronic glucose exposure also reduced cell surface insulin binding by 30% via an apparent decrease in receptor affinity, and this effect was associated with a comparable rightward shift in the insulin-glucose transport dose-response curve. In other studies, persistent stimulation with 15 nM insulin also decreased maximally stimulated glucose transport activity, which was independent and additive to the regulatory effect of glucose. Moreover, glucose and insulin-induced insulin resistance via different mechanisms. Glucose (25 mM) reduced the number of cellular glucose transporter proteins by 84% and levels of GLUT1 transporter mRNA by 50% (whether normalized to total RNA or CHO-B mRNA). In contrast, chronic insulin exposure led to a 2.1-fold increase in GLUT1 mRNA but did not alter cellular levels of transporter protein. Cotreatment with glucose prevented the insulin-induced rise in GLUT1 mRNA. BC3H1 cells did not express GLUT4 mRNA that encodes the major transporter isoform in skeletal muscle. In conclusion, in BC3H1 myocytes 1) glucose diminished insulin sensitivity by decreasing insulin receptor binding affinity and decreased basal and maximally insulin-stimulated glucose transport rates via cellular depletion of glucose transporters and suppression of GLUT1 mRNA; 2) chronic insulin exposure exerted an independent and additive effect to reduce maximal transport activity; however, insulin increased levels of GLUT1 mRNA and did not alter the cellular content of glucose transporters; and 3) although BC3H1 cells are commonly used as a model for skeletal muscle, studies examining glucose transport should be interpreted cautiously due to the absence of GLUT4 expression. Nevertheless, the data generally support the idea that, in non-insulin-dependent diabetes mellitus, hyperglycemia and hyperinsulinemia can induce or exacerbate insulin resistance in target tissues.     

Hypoxia regulates osteoblast gene expression.

Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-beta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor TGF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators.     

人前脑啡肽原基因修饰人骨髓间充质干细胞系的构建

目的 构建人前脑啡肽原(PENK)基因修饰的并可稳定分泌脑啡肽蛋白的人骨髓间充质干细胞系(hMSCs).方法 采用脂质体法将PENK基因逆转录病毒载体质粒(pBABE-PENK)转染至Phoenix-293T细胞,收集病毒上清液感染hMSCs细胞,经过嘌呤霉素筛选得到稳定表达PENK基因的hMSCs细胞株(hMSC-PENK细胞).以转染空载体细胞作为对照,即hMSC-pBABE细胞.采用RT-PCR法检测PENK mRNA的表达,免疫荧光法测定亮氨酸脑啡肽(LEK)的表达,ELISA法测定细胞培养上清液LEK浓度.结果 与hMSCs细胞和hMSC-pBABE细胞比较,hMSC-PENK细胞PENK mRNA和LEK表达上调,细胞培养上清液中LEK浓度升高(P＜0.05或0.01).结论 PENK基因修饰的hMSCs可表达PENK基因并分泌脑啡肽蛋白,成功构建了稳定分泌镇痛物质的细胞系.     

Region-specific constitutive gene expression in the adult porcine meniscus.

The knee meniscus exhibits extensive spatial variations in native healing capacity, biochemical composition, and cell morphology that suggest the existence of distinct phenotypes for meniscus cells. Constitutive gene expression levels of appropriate extracellular matrix proteins may serve as useful molecular markers of cellular phenotypes; however, relatively little is known of variations in the gene expression for meniscus cells of different regions of the tissue. The objective of the present study was to evaluate constitutive differences between radial inner and outer regions in gene expression for extracellular matrix proteins relevant to the meniscus. A secondary objective was to determine if these region-specific differences in gene expression are maintained after periods of monolayer culture. The innermost regions of the meniscus were found to constitutively express higher mRNA levels for proteins highly expressed in articular cartilage, including aggrecan, type II collagen, and NOS2. In contrast, the outer meniscus was found to contain higher gene expression for proteins associated with fibrous tissues including type I collagen, and the proteases MMP2 and MMP3. Isolated inner and outer meniscus cells maintained these region-specific gene expression patterns for collagens and proteoglycans during short-term monolayer culture. The results provide new information that suggests the utility of constitutive gene expression levels as molecular markers to distinguish tissue and cells of the inner and outer meniscus.     

Short-term hypothyroidism and vasopressin gene expression in the rat.

Hypothyroidism is associated with abnormalities in renal water handling, which include a delay in excretion of an acute water load, decreased urinary concentrating ability, and increased urine volume. In the present study, we investigated the role of vasopressin in aminotriazole-induced hypothyroidism by measuring vasopressin concentration in the plasma and pituitary along with vasopressin mRNA levels in the hypothalamus. After 5 weeks of aminotriazole treatment, L-thyroxine levels were significantly lower in the experimental animals (122 +/- 8 v 26 +/- 1 nmol/L [9.5 +/- 0.6 v 2.0 +/- 0.1 micrograms/dL]; P less than 0.001). Serum sodium (148 +/- 0.5 v 144 +/- 1.2 mmol/L [mEq/L]; P less than 0.01), and plasma osmolality (311 +/- 2.5 v 304 +/- 1.8 mmol/kg [mOsm/kg] H2O; P less than 0.05) were also lower in the experimental animals. There were no differences in plasma (1.9 +/- 0.4 v 1.5 +/- 0.2 pg/mL) or pituitary (1.5 +/- 0.4 v 1.5 +/- 0.2 microgram/pituitary) vasopressin levels. In addition, steady-state vasopressin mRNA levels were not different between the two groups (1,286 +/- 210 v 1,093 +/- 138 pg/hypothalamus). One week of L-thyroxine replacement resulted in significant increases in serum thyroxine levels without changes in the other variables measured. These results indicate that short-term hypothyroidism, which has been shown to exert substantial effects on renal function, causes only a modest central alteration in the plasma vasopressin-osmolality relationship, which occurs in the absence of detectable changes in vasopressin synthesis.     

Developmental expression of the neurotensin gene in the rat liver.

OBJECTIVE: This study determined whether the neurotensin gene is expressed during early development of the liver. SUMMARY BACKGROUND DATA: Neurotensin (NT), a gut tridecapeptide localized mainly to the distal small bowel and brain of adults, is an important hormone regulating gut motility, secretion and mucosal growth. Expression of NT peptide and the gene is found in fibrolamellar hepatocarcinomas, a variant of hepatocellular carcinoma, but not in the normal adult liver. METHODS: Northern and in situ hybridization techniques were used to determine expression of the neurotensin gene (NT/N) in the normal developing liver. RESULTS: NT/N is expressed in the fetal and early postnatal rat liver, but expression is repressed in the liver of the adult. In situ hybridization confirms the authors' Northern data and demonstrates a random distribution of NT/N expression in the fetal and 3-day postnatal liver. CONCLUSIONS: The authors conclude from this study that NT/N is expressed during early development of the rat liver with subsequent repression in the adult. NT/N may be reexpressed with malignant transformation of the liver.