Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.

Using U0126 to dissect the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade in the regulation of gene expression by endothelin-1 in cardiac myocytes

The hypertrophic agonist endothelin-1 rapidly but transiently activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade (and other signalling pathways) in cardiac myocytes, but the events linking this to hypertrophy are not understood. Using Affymetrix rat U34A microarrays, we identified the short-term (2-4 h) changes in gene expression induced in neonatal myocytes by endothelin-1 alone or in combination with the ERK1/2 cascade inhibitor, U0126. Expression of 15 genes was significantly changed by U0126 alone, and expression of an additional 78 genes was significantly changed by endothelin-1. Of the genes upregulated by U0126, four are classically induced through the aryl hydrocarbon receptor (AhR) by dioxins suggesting that U0126 activates the xenobiotic response element in cardiac myocytes potentially independently of effects on ERK1/2 signalling. The 78 genes showing altered expression with endothelin-1 formed five clusters: (i) three clusters showing upregulation by endothelin-1 according to time course (4 h > 2 h; 2 h > 4 h; 2 h approximately 4 h) with at least partial inhibition by U0126; (ii) a cluster of 11 genes upregulated by endothelin-1 but unaffected by U0126 suggesting regulation through signalling pathways other than ERK1/2; (iii) a cluster of six genes downregulated by endothelin-1 with attenuation by U0126. Thus, U0126 apparently activates the AhR in cardiac myocytes (which must be taken into account in protracted studies), but careful analysis allows identification of genes potentially regulated acutely via the ERK1/2 cascade. Our data suggest that the majority of changes in gene expression induced by endothelin-1 are mediated by the ERK1/2 cascade.

Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca(2+) and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca(2+) on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca(2+) imaging) methods. HCs exhibit a time- and voltage-dependent current (I(hc)), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca(2+)](o) from 20 nM to 2 μM reversibly decreases I(hc), decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca(2+)](o) modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I(hc) and modifies its kinetics. Rapid lowering of [Ca(2+)](o) from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I(hc) that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca(2+)](i). A step increase of [Ca(2+)](i) evoked by photorelease of Ca(2+) early during a depolarizing pulse led to a transient decrease of I(hc) superimposed on a growing outward I(hc); a step decrease of [Ca(2+)](i) elicited by photoactivation of a Ca(2+) scavenger provoked a transient increase in I(hc). Hence, it is tempting to assume that Ca(2+) exerts a direct effect on Cx45 hemichannels.

Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain.

Previous studies imply that the intracellular domain of Notch1 must translocate to the nucleus for its activity. In this study, we demonstrate that a mNotch1 mutant protein that lacks its extracellular domain but retains its membrane-spanning region becomes proteolytically processed on its intracellular surface and, as a result, the activated intracellular domain (mNotchIC) is released and can move to the nucleus. Proteolytic cleavage at an intracellular site is blocked by protease inhibitors. Intracellular cleavage is not seen in cells transfected with an inactive variant, which includes the extracellular lin-Notch-glp repeats. Collectively, the studies presented here support the model that mNotch1 is proteolytically processed and the cleavage product is translocated to the nucleus for mNotch1 signal transduction.

Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids.

It has been suggested that transepithelial gradients of short-chain fatty acids (SCFAs; the major anions in the colonic lumen) generate pH gradients across the colonic epithelium. Quantitative confocal microscopy was used to study extracellular pH in mouse distal colon with intact epithelial architecture, by superfusing tissue with carboxy SNARF-1 (a pH-sensitive fluorescent dye). Results demonstrate extracellular pH regulation in two separate microdomains surrounding colonic crypts: the crypt lumen and the subepithelial tissue adjacent to crypt colonocytes. Apical superfusion with (i) a poorly metabolized SCFA (isobutyrate), (ii) an avidly metabolized SCFA (n-butyrate), or (iii) a physiologic mixture of acetate/propionate/n-butyrate produced similar results: alkalinization of the crypt lumen and acidification of subepithelial tissue. Effects were (i) dependent on the presence and orientation of a transepithelial SCFA gradient, (ii) not observed with gluconate substitution, and (iii) required activation of sustained vectorial acid/base transport by SCFAs. Results suggest that the crypt lumen functions as a pH microdomain due to slow mixing with bulk superfusates and that crypts contribute significant buffering capacity to the lumen. In conclusion, physiologic SCFA gradients cause polarized extracellular pH regulation because epithelial architecture and vectorial transport synergize to establish regulated microenvironments.

Gene expression down-regulation in CD90(+) prostate tumor-associated stromal cells involves potential organ-specific genes

Background: The prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. The tumor-associated stroma is marked by increased expression of CD90/THYI. Isolation and characterization of these stromal cells could provide valuable insight into the biology of the tumor microenvironment. Methods: Prostate CD90(+) stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between histologically normal and tumor-associated stromal cells. For comparison, stromal cells were also isolated and analyzed from the urinary bladder. Results: The tumor-associated stromal cells were found to have decreased expression of genes involved in smooth muscle differentiation, and those detected in prostate but not bladder. Other differential expression between the stromal cell types included that of the CXC-chemokine genes. Conclusion: CD90(+) prostate tumor-associated stromal cells differed from their normal counterpart in expression of multiple genes, some of which are potentially involved in organ development.

ACE as a Mechanosensor to Shear Stress Influences the Control of Its Own Regulation via Phosphorylation of Cytoplasmic Ser(1270)

Objectives: We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser(1270) are involved in shear-stress (SS)-induced downregulation of the enzyme. Methods and Results: Western blotting analysis showed that SS (18 h, 15 dyn/cm(2)) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra-or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser(1270) compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor. Conclusions: ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser(1270), consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser(1270).

Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for CA-binding protein (GABP)

Within steroid receptor heterocomplexes the large tetraticopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (HspSO) and act coordinately with HspSO to modulate receptor activity. The reversible nature of the interaction between the immunophilins and HspSO suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a fi-kilobase (kb) 5 ' -flanking region of the human gene and demonstrated that a similar to 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GAFF is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Regulation of xylanase in Aspergillus phoenicis: a physiological and molecular approach

Microbial xylanolytic enzymes have a promising biotechnological potential, and are extensively applied in industries. In this study, induction of xylanolytic activity was examined in Aspergillus phoenicis. Xylanase activity induced by xylan, xylose or beta-methylxyloside was predominantly extracellular (93-97%). Addition of 1% glucose to media supplemented with xylan or xylose repressed xylanase production. Glucose repression was alleviated by addition of cAMP or dibutyryl-cAMP. These physiological observations were supported by a Northern analysis using part of the xylanase gene ApXLN as a probe. Gene transcription was shown to be induced by xylan, xylose, and beta-methylxyloside, and was repressed by the addition of 1% glucose. Glucose repression was partially relieved by addition of cAMP or dibutyryl cAMP.

Extracellular calcium stimulates Na+-dependent putrescine uptake in B16 melanoma cells

The regulation of putrescine transport in difluoromethylornithine-treated B16 melanoma cells by extracellular Ca2+ has been investigated. It was found that physiological concentrations of Ca2+ were essential for optimum uptake of putrescine and spermidine. Mg2+, albeit at higher concentrations, also could potentiate polyamine transport. The maximum rate of putrescine uptake increased from 1698 +/-: 67 pmol/min/mg DNA in the absence of Ca2+ to 3100 +/- 98 pmol/min/mg DNA in the presence of 0.5 mM Ca2+. There was no change in K-m. While Ca2+ enhanced transport of both putrescine and spermidine it did not affect the uptake of deoxyglucose, thymidine or leucine. Putrescine did not alter Ca2+ fluxes suggesting that the two cations do not share a common transport system. The effects of Ca2+ on putrescine uptake appeared to be mediated extracellularly firstly because Ca2+ did not potentiate putrescine uptake in the presence of A23187 and secondly, because the effects of Ca2+ were completely inhibited by the lanthanide Tb3+, which binds to calcium-dependent proteins and does not readily cross biological membranes. Ca2+ did not affect putrescine transport in the absence of extracellular Na+. Moreover, the rate of putrescine uptake in the absence of Ca2+ was similar to that in the absence of extracellular Na+. The results from this study indicate that polyamine transport is stimulated by extracellular Ca2+ and suggest that Ca2+ is required for activity of the Na+-dependent transporter only. This transporter appears to possess a regulatory binding site for divalent cations. (C) 1997 Elsevier Science Ltd.

Down-regulation of the amyloid protein precursor of Alzheimer's disease by antisense oligonucleotides reduces neuronal adhesion to specific substrata

The hallmark of Alzheimer's disease is the cerebral deposition of amyloid which is derived from the amyloid precursor protein (APP). The function of APP is unknown but there is increasing evidence for the role of APP in cell-cell and/or cell-matrix interactions. Primary cultures of murine neurons were treated with antisense oligonucleotides to down-regulate APP. This paper presents evidence that APP mediates a substrate-specific interaction between neurons and extracellular matrix components collagen type I, laminin and heparan sulphate proteoglycan but not fibronectin or poly-L-lysine. It remains to be determined whether this effect is the direct result of APP-matrix interactions, or whether an intermediary pathway is involved. (C) 1997 Elsevier Science B.V.

Dorsal raphe nucleus regulation of a panic-like defensive behavior evoked by chemical stimulation of the rat dorsal periaqueductal gray matter

Electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) evokes escape, a defensive behavior that has been related to panic attacks. Injection of 5-HT(1A) or 5-HT(2A) receptor agonists into this midbrain area inhibits this response. It has been proposed that the impairment of 5-HT mechanisms controlling escape at the level of the DPAG may underlie the susceptibility to panic attacks that characterizes the panic disorder. In this study we evaluated the effects of the pharmacological manipulation of the dorsal raphe nucleus (DRN), which are the main source of 5-HT input to the DPAG, on the escape response evoked in rats by the intra-DPAG injection of the nitric oxide donor SIN-1. The results showed that DRN administration of the 5-HT(1A) receptor agonist 8-OH-DPAT which inhibits the activity of 5-HT neurons favored the expression of escape induced by SIN-1. Intra-DRN injection of the excitatory amino acid kainic acid or the 5-HT(1A) receptor antagonist WAY-100635 did not change escape expression. However, both compounds fully blocked the escape reaction generated by intra-DPAG injection of the excitatory amino acid D,L-homocysteic acid (DLH). Overall, the results indicate that 5-HT neurons in the DRN exert a bidirectional control upon escape behavior generated by the DPAG. Taking into account the effect of WAY-100635 on DLH-induced escape, they also strengthen the view that DRN 5-HT(1A) autoreceptors are under tonic inhibitory influence by 5-HT. (C) 2010 Elsevier B.V. All rights reserved.

HYPOTHALAMIC COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT AND CORTICOTROPHIN RELEASING FACTOR NEURONS ARE STIMULATED BY EXTRACELLULAR VOLUME AND OSMOTIC CHANGES

Several studies suggest that hypothalamic cocaine- and amphetamine-regulated transcript (CART) may interact with the hypothalamic-pituitary-adrenal (HPA) axis in the control of neuroendocrine function and may also participate in cardiovascular regulation. Therefore, this study aimed to evaluate, in experimental models of isotonic (I-EVE) and hypertonic (H-EVE) extracellular volume expansion and water deprivation (WD), the activation of CART- and corticotrophin releasing factor (CRF)-immunoreactive neurons, as well as the relative expression of CART and CRF mRNAs in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Both H-EVE (0.30M NaCl, 2mL/100g of body weight, in 1 minute) and 24 hours of WD significantly increased plasma sodium concentrations, producing, respectively, either an increase or a decrease in extracellular volume. I-EVE (0.15M NaCl, 2mL/100g of body weight, in 1 minute) evoked a significant increase in the circulating volume accompanied by unaltered plasma concentrations of sodium. CART-expressing neurons of both magnocellular and parvocellular hypothalamic divisions were activated to produce Fos in response to H-EVE but not in response to I-EVE. Furthermore, increased expression of CART mRNA was found in the PVN of H-EVE but not I-EVE rats. These data show for the first time that EVE not only activates hypothalamic CRF neurons but also increases CRF mRNA expression in the PVN. In contrast, WD increases the number of CART-immunoreactive neurons activated to produce Fos in the PVN and SON but does not change the number of neurons double labeled for Fos and CRF or expression of CRF mRNA in the PVN. These findings provided new insights into the participation of CART in diverse processes within the PVN and SON, including its possible involvement in activation of the HPA axis and cardiovascular regulation in response to changes in extracellular volume and osmolality. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Transcription of the Neurospora crassa 70-kDa class heat shock protein genes is modulated in response to extracellular pH changes

Heat shock proteins belong to a conserved superfamily of molecular chaperones found in prokaryotes and eukaryotes. These proteins are linked to a myriad of physiological functions. In this study, we show that the N. crassa hsp70-1 (NCU09602.3) and hsp70-2 (NCU08693.3) genes are preferentially expressed in an acidic milieu after 15 h of cell growth in sufficient phosphate at 30A degrees C. No significant accumulation of these transcripts was detected at alkaline pH values. Both genes accumulated to a high level in mycelia that were incubated for 1 h at 45A degrees C, regardless of the phosphate concentration and extracellular pH changes. Transcription of the hsp70-1 and hsp70-2 genes was dependent on the pacC (+) background in mycelia cultured under optimal growth conditions or at 45A degrees C. The pacC gene encodes a Zn-finger transcription factor that is involved in the regulation of gene expression by pH. Heat shock induction of these two hsp genes in mycelia incubated in low-phosphate medium was almost not altered in the nuc-1 (-) background under both acidic and alkaline pH conditions. The NUC-1 transcriptional regulator is involved in the derepression of nucleases, phosphatases, and transporters that are necessary for fulfilling the cell`s phosphate requirements. Transcription of the hsp70-3 (NCU01499.3) gene followed a different pattern of induction-the gene was depressed under insufficient phosphate conditions but was apparently unaffected by alkalinization of the culture medium. Moreover, this gene was not induced by heat shock. These results reveal novel aspects of the heat-sensing network of N. crassa.