Expression Of Early Response Genes Research Articles

Regulation of eotaxin gene expression by TNF-alpha and IL-4 through mRNA stabilization: involvement of the RNA-binding protein HuR.

During inflammatory responses, a major posttranscriptional regulation of early response and inflammatory gene expression occurs through modulation of mRNA turnover. We report that two potent inducers of the CC chemokine eotaxin, TNF-alpha and IL-4, regulate its production in airway epithelial cells by increasing eotaxin mRNA stability. In experiments using the transcriptional inhibitor actinomycin D, eotaxin mRNA half-life was significantly prolonged by cell stimulation with TNF-alpha or IL-4, with the combination of the two cytokines being the most effective in extending the mRNA half-life. Involvement of the eotaxin 3' untranslated region in the mRNA-stabilizing effect was tested by transient transfection of a construct expressing a chimeric transcript carrying a serum-inducible beta-globin reporter linked to the eotaxin 3' untranslated region. The half-life of the chimeric mRNA was markedly increased in cells stimulated with TNF-alpha and IL-4. Evidence that the mRNA-stabilizing protein HuR participated in the cytokine effect was obtained: first, HuR presence in the cytoplasm, believed to be required for HuR-mediated mRNA stabilization, increased in both transformed (BEAS-2B cell line) and primary bronchial epithelial cells following treatment with TNF-alpha and IL-4. Second, endogenous eotaxin mRNA was found to bind to HuR in vivo, as detected by immunoprecipitation of HuR-containing messenger ribonucleoprotein complexes followed by real-time RT-PCR analysis; such association increased after cell treatment with TNF-alpha and IL-4. Third, overexpression of HuR in BEAS-2B cells significantly increased the expression of eotaxin mRNA and protein. Our findings implicate mRNA stabilization in the cytokine-mediated increase in eotaxin expression and strongly suggest a role for HuR in this effect.

Expression of early response genes, Fra1 and Fra2, by 17β-Estradiol in the ovariectomized mouse uterus

Objective: The early response genes induced by 17β-estradiol(E2) include the important regulatory genes, such as transcription factors. The aim of this study was to investigate the early response gene induced by E2 in the ovariectomized(Ovx) mouse uterus. Design: Experimental laboratory study using a mouse model. Materials and Methods: The Ovx mice were treated with a single s.c injection of E2(300ng/0.1ml/mouse) dissolved in sesame oil, while the control mice received oil only. They were given the pure antiestrogen ICI 182,780(500ng/mouse) 30min before the injection of E2. The Ovx mice were killed at 2, 4, 6 and 12 hr after the injection, and their uteri were collected for RNA extraction. The expression of the 15 transcription factors was examined by reverse transcription-polymerase chain reaction(RT-PCR).The localization of these protein was investigated by immunohistochemistry. Results: The expression patterns of genes were divided into four groups: Group I, early response genes include Fra1, Fra2, CREB2 and GATA5; Group II, response maintained genes include CREB1, ATF1, GLI3 and E2F3; Group III, late response genes include E2F1; Group IV, down regulated genes include GLI1, GLI2, E2F5, GATA2, GATA3 and GATA6. Among these early resposne genes, the Fra1 and Fra2 were selected for protein localization. The levels of Fra1 mRNA peaked 2.3-fold at 4 hr, the levels of Fra2 mRNA peaked 3.2-fold at 2 hr compared with the control values, respectively and these mRNA levels returned to basal levels at 12 hr. In addition, The ICI 182,780 clearly blocked the effect of E2. The expression of Fra1 and Fra2 protein was stronger in the myometrium than in other regions. Conclusion: The results of this study suggest that expression of the Fra1 and Fra2 mRNA is up-regulated by E2 and activated via an estrogen nuclear receptor in the mouse uterus and these protein may play an important role in mediating the contraction and relaxation of the uterine smooth muscle, but the functional significance of these protein in the myometrium is not clear.

Stimulation of Fos- and Jun-related genes during distraction osteogenesis.

Bone cells respond to mechanical stimulation by gene expression. The molecular events involved in the translation of mechanical stimulation into cell proliferation and bone formation are not yet well understood. We looked for the expression of early-response genes of the AP-1 transcription factor complex in an in vivo bone regeneration system subjected to mechanical forces because these genes were found to be related to mechanotransduction and important for bone development. Sheep maxillary bone was distracted daily for 15 days. c-Jun and c-Fos were evaluated by Northern blotting analysis and immunohistochemistry in biopsy specimens removed at 8 and 15 days and were compared with post-osteotomy but not distracted repair tissue. Elevated levels of c-Jun and c-Fos mRNA were found after 8 days of distraction. Likewise, mesenchyme-like and fibroblast-like cells composing the 8-day distracted regeneration tissue showed increases in the intensity of immunostaining compared to cells in the corresponding non-distracted fracture repair tissue. After 15 days of distraction, when bone trabeculae start to form distally and proximally in the distracted regeneration tissue, mostly preosteoblasts and osteoblasts retained c-Fos and c-Jun immunoreactivity, similar to bone-associated cells in control non-distracted fracture repair tissue. We propose that the elevated expression of c-Jun and c-Fos is related to mechanical stimulation in this in vivo bone regeneration system.

Region-specific changes in immediate early gene expression in response to sleep deprivation and recovery sleep in the mouse brain

Previous studies have documented changes in expression of the immediate early gene (IEG) c-fos and Fos protein in the brain between sleep and wakefulness. Such expression differences implicate changes in transcriptional regulation across behavioral states and suggest that other transcription factors may also be affected. In the current study, we examined the expression of seven fos/jun family member mRNAs (c-fos, fosB, fos related antigen (fra)1, fra-2, junB, c-jun, and junD) and three other IEG mRNAs (egr-1, egr-3, and nur77) in mouse brain following short-term (6 h) sleep deprivation (SD) and 4 h recovery sleep (RS) after SD. Gene expression was quantified in seven brain regions by real-time reverse transcription–polymerase chain reaction (RT-PCR). Multivariate analysis of variance revealed statistically significant variation in cerebral cortex, basal forebrain, thalamus and cerebellum. Levels of c-fos and fosB mRNA were elevated during SD in all four of these brain regions. In the cerebral cortex, junB mRNA was also elevated during SD whereas, in the basal forebrain, fra-1 and fra-2 mRNA levels increased in this condition. During RS, the only IEG mRNA to undergo significant increase was fra-2 in the cortex. C-jun and junD mRNAs were invariant across experimental conditions. These results indicate that the expression of fos/jun family members is diverse during SD. Among other IEGs, nur77 mRNA expression across conditions was similar to c-fos and fosB, egr-1 mRNA was elevated during SD in the cortex and basal forebrain, and egr-3 mRNA was elevated in the cortex during both SD and RS. The similarity of fosB and nur77 expression to c-fos expression indicates that these genes might also be useful markers of functional activity. Along with our previous results, the increased levels of fra-2 and egr-3 mRNAs during RS reported here suggest that increased mRNA expression during sleep is rare and may be anatomically restricted.

Lead-induced cell signaling cascades in GT1–7 cells

The effects of lead on the signal transduction pathways that may be involved in the release of gonadotropin-releasing hormone (GnRH) from neurons in the hypothalamus have not been well defined. Using the GT1–7 cell line, an in vitro model for GnRH-secreting neurons, we examined signal transduction pathways directly affected by lead. We found that lead-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2), as well as p90 RSK and cAMP response element-binding protein (CREB), but did not induce IκB degradation. MEK1/2 inhibitor (PD98059) suppressed lead-induced ERK and p90 RSK activation. Neither PKC inhibitors (Go6983, Go6976) nor CaMKII inhibitor (KN-62) had a pronounced effect on lead-induced ERK1 and ERK2 phosphorylation. However, MEK1/2 inhibitor, CaMKII inhibitor, and PKC inhibitor significantly suppressed lead-induced CREB phosphorylation. These results indicate that lead-activated PKC, CaMKII and MEK/ERK/p90 RSK pathways simultaneously, all of which contributed to CREB phosphorylation. Our results also indicate that lead-induced p90 RSK and CREB activation does not alter expression of early response genes like c- fos. We conclude that lead activates PKC, CaMKII or MEK-ERK-p90 RSK pathways in GT1–7 cells, leading to CREB phosphorylation and modulation of gene expression.

Vascular smooth muscle cell activation by C-reactive protein.

C-reactive protein (CRP) is an important cardiovascular risk factor. Although the role of CRP has been implicated in atherogenesis, its direct effects on vascular cells are poorly defined. We investigated the responses to CRP in vascular smooth muscle cells (VSMC). The present study shows that CRP induces parallel activation of the redox-responsive transcription factors NF-kappa B (NF-kappaB) and AP-1 and increases the activity of the MAP kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38MAPK, in VSMC. C-reactive protein increased the expression of early response genes, c-fos and c-jun and inflammatory genes, monocyte chemoattractant peptide (MCP-1) and interleukin-6 (IL-6). When VSMC were incubated with CRP, the inducible nitric oxide synthase (iNOS) promoter was activated. CRP alone was a weak inducer of NO production in VSMC as measured by determining nitrite levels, and interferon-gamma alone was totally ineffective, whereas CRP plus interferon-gamma was a powerful stimulus. This synergy for NO production corresponded to the results of iNOS mRNA expression analyzed by Northern blotting. The NF-kappaB activation caused by CRP was inhibited by 15-deoxy-12,14-prostaglandin J2 and the PPARgamma activators, rosiglitazone and pioglitazone. Fluvastatin and cerivastatin also reduced the activation of NF-kappaB by CRP. CRP causes NF-kappaB activation which could lead to the induction of MCP-1, IL-6, and iNOS gene expression. CRP also activates the MAPK-->c-Fos/cJun-->AP-1 pathway. Thus, CRP may play a role in atherogenesis by activating VSMC.

Induction of c-fos expression by nicotine in human periodontal ligament fibroblasts is related to cellular thiol levels.

Cigarette smoking is associated with increased incidence of periodontal disease and poor response to periodontal therapy. Several studies have shown the detrimental effects of nicotine on periodontal tissue. To investigate the molecular toxicological implications of cigarette smoking on periodontal tissue, expression of c-fos early stress response gene was examined in human periodontal ligament fibroblasts (PDLFs) after exposure to nicotine. The exposure of quiescent human PDLFs to nicotine resulted in the induction of c-fos mRNA expression. The levels of the c-fos mRNAs increased about 2.5 and 4.8-fold after exposure to 2.5 mm and 10 mm nicotine for 2 h, respectively. Moreover, the peak of c-fos mRNA levels induced by nicotine was 5 mm at 2-h incubation period. Kinetic investigations of c-fos mRNA expression in nicotine-treated cells revealed a rapid accumulation of the transcript, a significant signal first detectable after 30 min of exposure. This increase was transient and the level of c-fos mRNAs returned rapidly to that of control cells by 8 h. To determine whether thiol levels were important in induction of c-fos by nicotine, we pretreated cells with the glutathione (GSH) precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Our results demonstrate that OTZ pretreatment decreased in c-fos mRNA level and BSO pretreatment enhanced in c-fos mRNA level after exposure to nicotine. In addition, nicotine significantly depleted intracellular GSH in a dose-dependent manner (P < 0.05). At a concentration of 5 mm and 20 mm, nicotine depleted about 22.2% and 56% of GSH, respectively. Taken together, c-fos gene expression might be one signal transduction pathway linked to the induction of early response genes by cigarette smoking. These results suggest that the nicotine-dependent stress-specific expression of the c-fos gene correlates with cellular thiol levels in human PDLFs.

Rice receptor for chitin oligosaccharide elicitor does not couple to heterotrimeric G‐protein: Elicitor responses of suspension cultured rice cells from Daikoku dwarf (d1) mutants lacking a functional G‐protein α‐subunit

We present evidence that the rice receptor for N‐acetylchitooligosaccharide elicitor does not couple to heterotrimeric G‐protein (G‐protein), one of the most important signal transducers from the cell surface to down‐stream effectors in various cellular responses of many organisms. Using mutant rice cells lacking functional G‐protein α‐subunit, cellular responses of suspension‐cultured rice cells derived from Daikoku dwarf (d1) mutants that were shown to contain mutations in the coding region of the G‐protein α‐subunit (Fujisawa et al. 1999) to N‐acetylchitooligosaccharide (oligochitin) elicitor were compared with those of the corresponding parent cell lines. All the elicitor‐induced cellular responses, such as medium alkalinization, generation of reactive oxygen species, expression of early responsive genes, PAL and PR genes, and production of phytoalexin, were basically identical in the mutant and wild type cell lines. Considering the reported presence of a single copy gene for the G‐protein α‐subunit in many plant species including rice, these results strongly support the above conclusion.

C-Fos expression in ouabain-treated vascular smooth muscle cells from rat aorta: evidence for an intracellular-sodium-mediated, calcium-independent mechanism.

In this study, we examined the effect of Na(+)-K(+) pump inhibition on the expression of early response genes in vascular smooth muscle cells (VSMC) as possible intermediates of the massive RNA synthesis and protection against apoptosis seen in ouabain-treated VSMC in our previous experiments. Incubation of VSMC with ouabain resulted in rapid induction of c-Fos protein expression with an approximately sixfold elevation after 2 h of incubation. c-Jun expression was increased by approximately fourfold after 12 h, whereas expression of activating transcription factor 2, cAMP/Ca(2+) response element binding protein (CREB)-1 and c-Myc was not altered. Markedly augmented c-Fos expression was also observed under Na(+)-K(+) pump inhibition in potassium-depleted medium. Na(+)-K(+) pump inhibition triggered c-Fos expression via elevation of the [Na(+)](i)/[K(+)](i) ratio. This conclusion follows from experiments showing the lack of effect of ouabain on c-Fos expression in high-potassium-low-sodium medium and from the comparison of dose responses of Na(+)-K(+) pump activity, [Na(+)](i) and [K(+)](i) content and c-Fos expression to ouabain. A fourfold increment of c-Fos mRNA was revealed 30 min following addition of ouabain to the incubation medium. At this time point, treatment with ouabain resulted in an approximately fourfold elevation of [Na(+)](i) but did not affect [K(+)](i). Augmented c-Fos expression was also observed under VSMC depolarization in high-potassium medium. Increments in both c-Fos expression and (45)Ca uptake in depolarized VSMC were abolished under inhibition of L-type Ca(2+) channels with 0.1 microM nicardipine. Ouabain did not affect the free [Ca(2+)](i) or the content of exchangeable [Ca(2+)](i). Ouabain-induced c-Fos expression was also insensitive to the presence of nicardipine and [Ca(2+)](o), as well as chelators of [Ca(2+)](o) (EGTA) and [Ca(2+)](i) (BAPTA). The effect of ouabain and serum on c-Fos expression was additive. In contrast to serum, however, ouabain failed to activate the Elk-1, serum response factor, CREB and activator protein-1 transcription factors identified within the c-Fos promoter. These results suggest that Na(+)-K(+) pump inhibition triggers c-Fos expression via [Na(+)](i)-sensitive [Ca(2+)](i)-independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter.

Secretory phospholipase A(2) induces delayed neuronal COX-2 expression compared with glutamate.

Agonists of the binding site for secretory phospholipase A(2) (sPLA(2)) potentiate glutamate-induced neuronal cell death in primary cell cultures and in vivo (Kolko et al. [1996] J. Biol. Chem. 271:32722; Kolko et al. [1999] Neurosci. Lett. 274:167]. Here, we tested the hypothesis that COX-2 expression participates in the brain response to sPLA(2). sPLA(2)-OS(2), a selective ligand of a neuronal sPLA(2)-binding site, was injected into the rat striatum, and early-response gene expression was monitored by in situ hybridization using (35)S-radiolabeled oligonucleotide probes and immunohistochemistry. An up-regulation of COX-2, c-fos, and c-jun, but not COX-1, was observed around the lesion as well as in the neocortex 4 hr after the injection. Hippocampal up-regulation of COX-2 was seen in dentate gyrus 8 hr after injection. When glutamate was injected, up-regulation of the early-response genes peaked after 2 hr. Our studies showed 1) that sPLA(2) selectively induced neuronal COX-2; 2) that this induction was delayed (4 hr after injection of sPLA(2)) compared with that elicited by glutamate (2 hr after injection), suggesting different signaling; and 3) that c-fos and c-jun were induced around the infarct area as soon as 2 hr after injection, but in other aspects followed a time course similar to that of COX-2. We conclude that sPLA(2) may modulate neuronal COX-2 expression through mechanisms that differ from those of glutamate-induced COX-2 expression.

Short-chain fatty acids and total parenteral nutrition affect intestinal gene expression.

The supplementation of total parenteral nutrition (TPN) formulas with short-chain fatty acids (SCFAs) increases glucose uptake and the expression of glucose transporters in parenterally fed animals. Several signals may be involved in intestinal adaptation; however, increased messenger RNA (mRNA) levels for proglucagon and several early-response genes, including c-myc and c-fos, are seen in animals receiving SCFA-supplemented TPN. Although the effects of a mixture of SCFAs are well documented, the relative contribution of individual SCFAs is unknown. Butyrate is a preferred fuel of colonocytes, with documented effects on cellular proliferation and gene expression. Accordingly, this study was undertaken to determine the relative role of butyrate in initiating an adaptive response in nonresected rats receiving TPN. Animals received standard TPN for 66 hours, followed by 6 hours of either standard TPN, TPN supplemented with a mixture of SCFAs (acetate, propionate, and butyrate, 60 mmol/L total), or TPN supplemented with butyrate alone (9 mmol/L). An oral control group was fed an elemental diet, similar in macronutrient content to the TPN, so that all animals received the same amount of energy daily. SCFAs increased ileal glucose transporter 2 (GLUT2) mRNA expression compared with the orally fed group. SCFAs also increased proglucagon mRNA expression compared with the TPN group. No changes in Na+K(+)-adenosine triphosphatase or early-response gene expression were found in this study. In a rat model of TPN, the use of 9 mmol/L butyrate did not have the same effect on GLUT2 and proglucagon expression as a 60-mmol/L mixture of SCFAs. This suggests that the effect of a mixture of SCFAs on intestinal gene expression is not butyrate specific.

Acute intrarenal infusion of ANG II does not stimulate immediate early gene expression in the kidney.

ANG II is capable of stimulating expression of immediate early genes such as egr-1 and c-fos in a variety of cultured cells, including cells of renal origin. To investigate whether ANG II can stimulate early growth response gene expression in vivo, we studied the effects of acute renal artery infusion of low-dose ANG II (2.5 ng small middle dot kg(-1) small middle dot min(-1)) or vehicle on the renal expression of c-fos and egr-1 genes in rats. ANG II infusion for 30 or 240 min decreased renal vascular conductance by approximately 13 and 8%, respectively, compared with the vehicle group. Expression of the early growth response genes c-fos and egr-1 was analyzed using Northern blot hybridization. No significant upregulation of c-fos or egr-1 mRNA levels was detected in rats that received ANG II for either 30 or 240 min, compared with the vehicle groups. We conclude that ANG II, at doses that cause significant physiological effects, does not increase the renal expression of c-fos or egr-1 genes over periods of up to 4 h in vivo.

Elliptically polarized magnetic fields do not alter immediate early response genes expression levels in human glioblastoma cells.

Expression of immediate early response genes such as c-fos, c-jun, and c-myc in response to 1-500 microT resultant (r) 60 Hz elliptically polarized (EP) magnetic fields (MFs), typical of environmental MFs polarization under overhead power lines, was analyzed in both at transcriptional and translational levels using human glioblastoma (T98G) cells. Pseudo synchronized T98G cells at G1 phase were exposed to EP-MFs (1, 20, 100, and 500 microTr) for up to 3 h, but produced no statistical difference (P>0.05) in the levels of expression ratio at both the transcriptional and translational levels at 30 min for c-fos and c-jun and at 180 min for c-myc after serum stimulation. In addition, exposure of T98G cells to linearly (vertical and horizontal) and/or circularly polarized MFs (500 microTr) produced no significant change (P>0.05) in the expression ratio at both transcriptional and post-transcriptional levels. Thus, there was no evidence that linearly or rotating polarized MFs enhanced early response gene expression in these studies. These results suggest that environmental MFs at 1-500 microT flux density are unlikely to induce carcinogenesis through a mechanism involving altered expression of the immediate early response genes.

Vascular smooth muscle cell activation by glycated albumin (Amadori adducts).

Nonenzymatic glycation is increased in diabetes. The role of advanced glycation end products has been implicated in many of the complications of diabetes, whereas the effects of early-glycation Amadori-modified proteins on vascular cells alone are poorly defined. In the present study, we show that glycated serum albumin (GSA) induces a parallel activation of the redox-responsive transcription factors (nuclear factor kappaB) and AP-1 and increases activity of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38 MAPK in vascular smooth muscle cells (VSMCs). GSA increased expression of early response genes, c-fos and c-jun, and inflammatory genes, monocyte chemoattractant peptide (MCP-1), and interleukin (IL)-6. These effects were comparable to bacterial lipopolysaccharide, tumor necrosis factor-alphaa, (TNF-alphaa), IL-1alphab, angiotensin II, epidermal growth factor, and the phorbol ester PMA. One of signaling pathways by which GSA activates VSMCs appears to be via nuclear factor kappaB activation, leading to induction of MCP-1 and IL-6 gene expression, comparable to the effects of lipopolysaccharide, TNF-alphaa, and IL-1alphab. Another signaling cascade by which GSA activates VSMCs is the ERK-->c-Fos-->AP-1 pathway, which may lead to stimulation of cell proliferation and migration. These effects are comparable to the effects of angiotensin II, epidermal growth factor, and PMA. Incubation of VSMCs with the antioxidant N-acetylcysteine suppressed GSA-elicited mRNA induction of MCP-1 and IL-6. Inhibition of p38 MAPK but not ERK caused attenuation of MCP-1 and IL-6 mRNA induction. Finally, GSA caused a significant stimulation of VSMC growth and migration. These findings suggest that GSA may play a role in diabetic atherogenesis by activating VSMCs, leading to induction of inflammatory mediators in the vessel wall, as well as proliferation and migration of VSMCs.

Early response gene expression during differentiation of cultured Leishmania donovani.

The promastigote form of the unicellular parasite, Leishmania donovani, must differentiate into the amastigote form to establish an infection in a mammalian host. Identification of genes whose expression changes during differentiation could help reveal mechanisms of Leishmania gene regulation and identify targets for controlling the diseases caused by this human pathogen. Two genomic clones were isolated, P9 that is more highly expressed in promastigotes than in axenic amastigotes and A14 that is preferentially expressed in axenic amastigotes. Analysis of the DNA sequences revealed open reading frames that would encode 55.5 kDa and 100 kDa proteins, respectively, with no homology to known proteins. The mRNA level for these genes during 24 h time courses of parasite differentiation in culture was compared to two genes known to be differentially expressed, c-lpk2 and mkk. Changes in RNA level occurred within 2 h for each gene and continued in advance of morphological changes. The expression levels of these four genes in axenic amastigotes correlated with results from animal-derived parasites.

Expression of the receptor tyrosine kinase KIT in mature beta-cells and in the pancreas in development.

In the pancreas, ligands of receptor tyrosine kinases (RTKs) are thought to be implicated in the development and function of the islets of Langerhans, which represent the endocrine part of the pancreas. In a previous study, we randomly screened by reverse transcriptase-polymerase chain reaction for RTKs expressed in the embryonic pancreas. One cDNA fragment that was cloned during this screen corresponded to the KIT receptor. The objective of the present study was to analyze the pattern of Kit expression in the pancreas. We demonstrated that Kit is expressed and functional in terms of signal transduction in the insulin-producing cell line INS-1. Indeed, upon treatment with the KIT ligand (KITL), the extracellular signal-regulated protein kinase was phosphorylated, and the expression of early responsive genes was induced. We also demonstrated that Kit mRNAs are present in fetal and adult rat islets. We next used mice that had integrated the lacZ reporter gene into the Kit locus. In these mice, beta-galactosidase (beta-gal) served as a convenient marker for expression of the endogenous Kit gene. Kit was found to be specifically transcribed in beta-cells (insulin-expressing cells), whereas no expression was found in other endocrine cell types or in the exocrine tissue. Interestingly, not all mature beta-cells expressed Kit, indicating that Kit is a marker of a subpopulation of beta-cells. Finally, by following beta-gal expression in the pancreas during fetal life, we found that at E14.5, Kit is expressed in both insulin- and glucagon-expressing cells present at that stage, and also in a specific cell population present in the epithelium that stained negative for endocrine markers. These data suggest that these Kit-positive/endocrine-negative cells could represent a subpopulation of endocrine cell precursors.

C-Fos oncogene regulator Elk-1 interacts with BRCA1 splice variants BRCA1a/1b and enhances BRCA1a/1b-mediated growth suppression in breast cancer cells.

Elk-1, a c-Fos protooncogene regulator, which belongs to the ETS-domain family of transcriptional factors, plays an important role in the induction of immediate early gene expression in response to a variety of extracellular signals. In this study, we demonstrate for the first time the in vitro and in vivo interaction of Elk-1 with BRCA1 splice variants BRCA1a and BRCA1b using GST-pull down assays, co-imunoprecipitations/Western blot analysis of cell extracts from breast cancer cells and mammalian two-hybrid assays. We have localized the BRCA1 interaction domain of Elk-1 protein to the conserved ETS domain, a motif involved in DNA binding and protein-protein interactions. We also observed binding of BRCA1 proteins to other ETS-domain transcription factors SAP1, ETS-1, ERG-2 and Fli-1 but not to Elk-1 splice variant DeltaElk-1 and c-Fos protooncogene. Both BRCA1a and BRCA1b splice variants function as growth suppressors of human breast cancer cells. Interestingly, our studies reveal that although both Elk-1 and SAP-1 are highly homologous members of a subfamily of ETS domain proteins called ternary complex factors, it is only Elk-1 but not SAP-1 that can augment the growth suppressive function of BRCA1a/1b proteins in breast cancer cells. Thus Elk-1 could be a potential downstream target of BRCA1 in its growth control pathway. Furthermore, we have observed inhibition of c-Fos promoter activity in BRCA1a transfected stable breast cancer cells and over expression of BRCA1a/1b attenuates MEK-induced SRE activation in vivo. These results demonstrate for the first time a link between the growth suppressive function of BRCA1a/1b proteins and signal transduction pathway involving Elk-1 protein. All these results taken together suggest that one of the mechanisms by which BRCA1a/1b proteins function as growth/tumor suppressors is through inhibition of the expression of Elk-1 target genes like c-Fos.

Carcinogenic metals and NF-kappaB activation.

Epidemiological and animal studies suggest that several metals and metal-containing compounds are potent mutagens and carcinogens. These metals include chromium, arsenic, vanadium, and nickel. During the last two decades, chemical and cellular studies have contributed enormously to our understanding of the mechanisms of metal-induced pathophysiological processes. Although each of these metals is unique in its mechanism of action, some common signaling molecules, such as reactive oxygen species (ROS), may be shared by many of these carcinogenic metals. New techniques are now available to reveal the mechanisms of carcinogenesis in precise molecular terms. In this review, we focused our attentions on metal-induced signal transduction pathways leading to the activation of NF-kappaB, a transcription factor governing the expression of most early response genes involved in a number of human diseases.

Protective Effect of Salvia miltiorrhiza on Angiotensin II-Induced Hypertrophic Responses in Neonatal Rat Cardiac Cells

The effect of the root of Salvia miltiorrhiza (SM) on angiotensin II (Ang II)-induced hypertrophic responses was examined in cultured neonatal rat cardiac cells (cardiomyocytes and non-cardio-myocytes). The methanol eluate fraction (SM2) of the water extract and the ethyl acetate-insoluble fraction (SM3) and its soluble fraction (SM4) partitioned from the methanol extract were prepared. Treatment with SM4 (5 - 80 μ g/ml), not SM2 and SM3, for 24 h produced dose-dependent cytotoxicity against cardiac cells relative to the reduction in viability and the morphological injury of cardiomyocytes. SM2 or SM3 in the absence of Ang II affected neither hyperplastic nor hypertrophic growth of both cell types. However, SM3 (40 μ g /ml) attenuated the positive chronotropic responsiveness of cardiomyocytes to Ang II (1 nM) stimulation, whereas Ang II-induced increase in non-cardiomyocyte number was decreased only by SM2 (40 μ g /ml) treatment. Furthermore, SM3 suppressed Ang II-induced enlargement of cell size by preceding Ang II-induced induction of immediate early response gene (c-jun) expression in cardiomyocytes, while SM2 decreased Ang II-induced DNA synthesis in non-cardiomyocytes. Moreover, three phenolic compounds and tanshinone IIA that differed quantitatively among three SM fractions were identified by reverse-phase high performance liquid chromatography. Thus, the present findings indicate that the root of SM is an effective inhibitor of Ang II action and has a plural effective constituent, which possess different pharmacological activities on Ang II-induced hypertrophy and hyperplasia in cultured neonatal rat cardiac cells.

Novel approaches to chemoprevention of skin cancer.

Protection against sun-induced damage leading to photocarcinogenesis in skin is a highly desirable goal. Among various strategies, chemopreventive approaches utilizing non-toxic agents to prevent the occurrence of precancerous lesions or their surrogate markers are potentially attractive. Epidemiological and experimental studies provide evidence that some naturally occurring chemical agents in the human diet can diminish cancer risk. Aside from water, tea is the most common beverage consumed worldwide. Black tea accounts for nearly 80% of total tea production. Black tea and green tea are derived from the same plant, Camelia sinensis. Green tea contains monomeric polyphenols known as flavanols and black tea contains dimeric flavanols and polymeric polyphenols known as theaflavins (TFs) and thearubigins (TRs). Over the past fifteen years our laboratory has been exploring the feasibility of using tea and its constitutents as an approach to skin cancer prevention. We demonstrated that green tea, black tea and constituent polyphenols protect against chemical- and ultraviolet B (UVB)-induced carcinogenesis and reduce the growth of established tumors in skin. We have also shown the efficacy of green and black tea extracts against UVB and psoralen + ultraviolet A (PUVA)-induced early damage in skin. Although PUVA is highly effective in treating certain skin diseases, careful follow-up studies of cohorts of patients have shown that similar to UVB, PUVA treatment increases the risk for cutaneous squamous cell carcinoma and melanoma. We have found that oral administration of a standardized green tea extract (SGTE) prior to and during treatment of SKH-1 mice diminished PUVA-induced skin hyperplasia and hyperkeratosis. SGTE-treatment also inhibited PUVA-induced accumulation of c-fos and p53 proteins and epithelial hyperproliferation. Both topical application and oral administration of SGTE after PUVA-treatment reduced skin inflammation and cell hyperproliferation. Topical application of SGTE to human skin prior to PUVA-treatment inhibited the delayed skin inflammatory response. Similarly, oral and topical administration of standardized black tea extract (SBTE) and its two major polyphenolic sub-fractions protect against UVB-induced erythema in SKH-1 mice. Furthermore, topical application of tea extracts to human volunteers protects against UVB-induced erythema. In summary, these studies indicate that tea extracts are effective in reducing UVB- and PUVA-mediated DNA damage, expression of early response genes and early inflammatory changes in skin. These studies verify a conceptual rationale for employing naturally occurring dietary constitutents as an approach to cancer chemoprevention.