Gummatous penile syphilis.

Syphilitic gumma involving the penis is a rare manifestation of tertiary syphilis. Only seventeen cases have been reported in the literature. It can mimic other diagnoses such as penile carcinoma. We report a case of a 56 year old male that had been sexually abstinent for over 10 years and presenting with a 4 cm painful penile lesion with clinically palpable bilateral inguinal nodes with no prior history of sexually transmitted diseases (STDs). A positron emission tomography-computed tomography scan identified the penile mass as being hypermetabolic and suspicious for penile carcinoma. Several inguinal and pelvic lymph nodes were also found to be suspicious for penile carcinoma. A penile biopsy was proposed and declined by the patient as he opted for a partial penectomy. The surgery was performed for diagnostic and palliative purposes. Histopathological studies revealed the presence of polymorphous, granulomatous, epitheloid inflammatory infiltrate with giant cells. Additional microbiologic testing confirmed the diagnosis of tertiary syphilis, presenting as gummatous syphillis associated with neurosyphilis. The patient was treated with intravenous penicillin and had adequate clinical clinical and serologic 12 months following treatment. Gummatous syphillis is a rare entity, but should be considered in the differential diagnosis of a penile lesion. To rule out this possibility, a biopsy should always be performed prior to invasive penis surgery.

Colostrum whey down-regulates the expression of early and late inflammatory response genes induced by Escherichia coli and Salmonella enterica Typhimurium components in intestinal epithelial cells.

Pathogenic invasion by Escherichia coli and Salmonellae remains a constant threat to the integrity of the intestinal epithelium and can rapidly induce inflammatory responses. At birth, colostrum consumption exerts numerous beneficial effects on the properties of intestinal epithelial cells and protects the gastrointestinal tract of newborns from pathogenic invasion. The present study aimed to investigate the effect of colostrum on the early and late inflammatory responses induced by pathogens. The short-term (2 h) and long-term (24 h) effects of exposure to heat-killed (HK) E. coli and Salmonella enterica Typhimurium on gene expression in the porcine intestinal epithelial cell (IPEC-J2) model were first evaluated by microarray and quantitative PCR analyses. Luciferase assays were performed using a NF-κB-luc reporter construct to investigate the effect of colostrum whey treatment on the activation of NF-κB induced by HK bacteria. Luciferase assays were also performed using NF-κB-luc, IL-8-luc and IL-6-luc reporter constructs in human colon adenocarcinoma Caco-2/15 cells exposed to dose-response stimulations with HK bacteria and colostrum whey. Bovine colostrum whey treatment decreased the expression of early and late inflammatory genes induced by HK bacteria in IPEC-J2, as well as the transcriptional activation of NF-κB-luc induced by HK bacteria. Unlike that with colostrum whey, treatment with other milk fractions failed to decrease the activation of NF-κB-luc induced by HK bacteria. Lastly, the reduction of the HK bacteria-induced activation of NF-κB-luc, IL-8-luc and IL-6-luc by colostrum whey was dose dependent. The results of the present study indicate that bovine colostrum may protect and preserve the integrity of the intestinal mucosal barrier in the host by controlling the expression levels of early and late inflammatory genes following invasion by enteric pathogens.

Higher circulating 4-hydroxynonenal-protein thioether adducts correlate with more severe diastolic dysfunction in spontaneously hypertensive rats.

OBJECTIVE: Accumulating evidence supports a role of 4-hydroxynonenal (4HNE) in oxidative-stress related diseases, but its specific contribution to disease development remains to be clarified. Further to our finding of high circulating 4HNE-protein thioether adducts (4HNE-P) in spontaneously hypertensive rats (SHRs), we aimed at correlating 4HNE-P with cardiac function and testing the impact of antioxidant therapy. MATERIALS AND METHODS: The lipoperoxidation inhibitor probucol (10 mg/kg/day) or vehicle (corn oil) were administered daily (i.p.) for 4 weeks in 18-week-old SHRs (9 rats/group). Cardiac functions were assessed by echocardiography and 4HNE-P by gas chromatography/mass spectrometry. RESULTS: Diastolic dysfunction worsened in SHRs receiving vehicle as reflected by changes (P < 0.05) in indexes of left ventricular relaxation (increased isovolumic relaxation time) and compliance (increased E-wave deceleration rate; EDR). Higher circulating 4HNE-P correlated with diastolic dysfunction (EDR: R(2) = 0.518; P < 0.001) and heart rate (R(2) = 0.225; P < 0.05). Probucol prevented the deterioration of diastolic function, while lowering the mean and median of circulating 4HNE-P by 21% and 35%, respectively. CONCLUSION: Collectively, these results support a role for 4HNE in the pathophysiological events linked to disease progression in SHRs.

Angiotensin II AT1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells.

The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT1R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca]c) and nuclear ([Ca]n) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca2+ and hAT1R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT1R and the absence of hAT2R in normal hVSMCs. Using 3-D imaging technique, hAT1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT1R or hAT1R-GFP overexpressing hVSMCs, Ang II (10(-9) and 10(-4) M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT1Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT1R. The Ang II induced increase of hAT1R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT1R and hAT1R-GFP decreased both basal cytosolic and nuclear Ca2+. In normal hVSMCs and low hAT1R-GFP overexpressing hVSMCs, Ang II (10(-15) to 10(-4) M) induced a dose-dependent sustained increase of [Ca]c and [Ca]n with an EC50 near 5 x 10(-11) and 5 x 10(-9) M, respectively. Our results suggest that hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT1R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT1R overexpression in hVSMCs modulates sustained [Ca]c and [Ca]n.

Effect of human recombinant leptin on lipid handling by fully differentiated Caco-2 cells.

It has been established that leptin displays a number of effects on peripheral tissues. We have investigated the effect of the hormone on lipid synthesis, apolipoprotein biogenesis and lipoprotein secretion in Caco-2 cells. Immunocytochemistry revealed the presence of leptin receptors (Ob-Rb) on the basolateral membrane. Incubation of cells with 200 nM leptin resulted in a decreased export of triglycerides in the basolateral medium without affecting monoglyceride, diglyceride and cholesterol ester lipid classes. It also significantly reduced the output of de novo-synthesized apolipoprotein (Apo)B-100 and ApoB-48 as well as that of newly formed chylomicrons and of low-density lipoproteins. It also enhanced that of ApoA-I, ApoA-IV and ApoE. Our results support the hypothesis that leptin can affect energy balance at the gut level by reducing lipid release into the circulation.

Role of specific CCAAT/enhancer-binding protein isoforms in intestinal epithelial cells.

Intestinal epithelial cells participate in the acute phase response in response to inflammation. We have shown that acute phase protein genes are induced during intestinal acute phase response, and that the CCAAT/enhancer binding protein family of transcription factors are involved. To address the role of specific C/EBP isoforms, we generated IEC-6 rat intestinal epithelial cell lines expressing different C/EBP isoforms, by retroviral infection. Overexpression of C/EBPalpha p30 and C/EBPdelta led to increases in C/EBPbeta LAP and C/EBPbeta LIP endogenous protein levels, as determined by electrophoretic mobility shift assays and Western blot. Inhibition of C/EBP activity with dominant negative C/EBPs (C/EBPbeta LIP, 3hF, 4hF) decreased glucocorticoid-, cAMP- and IL-1 responsiveness of the endogenous haptoglobin gene, while overexpression of each C/EBP isoform increased the responsiveness to these regulators. In contrast, dominant negative C/EBPs or C/EBP isoforms did not alter the expression of alpha-acid glycoprotein in response to dexamethasone and of C/EBPbeta and C/EBPdelta in response to various regulators as assessed by Northern blot. These data show that the three C/EBP isoforms are involved in the regulation of haptoglobin and that C/EBPbeta, C/EBPdelta, and alpha-acid glycoprotein expression are not induced by C/EBP isoforms in contrast to other cell types. C/EBPbeta LAP-expressing cells showed an inhibition of cell growth characterized by a delay in p27(Kip1) decrease in response to serum and a decrease in cyclin D isoforms and cyclin E protein levels. Finally, C/EBP isoforms interact with the E2F4 transcription factor. Thus, specific C/EBP isoforms are involved in the differential expression of acute phase protein genes in response to hormones and cytokines. Furthermore, C/EBP isoforms may play a role in the control of cell cycle progression.

G-protein-independent activation of Tyk2 by the platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activity through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed that PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the monocytic cell lines U937 and MonoMac-1. PAF-initiated Tyk2 phosphorylation was also observed in COS-7 cells transiently transfected with the human PAF receptor (PAFR) and Tyk2 cDNAs. In addition, we found that Tyk2 co-immunoprecipitated and co-localized with PAFR, independently of ligand binding. Deletion mutants of Tyk2 indicated that the N terminus of the kinase was important for the binding to PAFR. Activation of Tyk2 was followed by a time-dependent 2-4-fold increase in the level of tyrosine phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 and a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In MonoMac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stimulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cells were transfected with PAFR and constructs containing PAFR promoter 1, coupled to the luciferase reporter gene, PAF induced a 3.6-fold increase in promoter activation in the presence of Tyk2. Finally, PAFR mutants that could not couple to G-proteins were found to effectively mediate Tyk2 activation and signaling. Taken together, these findings suggest an important role for the Janus kinase/STAT pathway in PAFR signaling, independent of G-proteins, and in the regulation of PAF receptor expression by its ligand.

Intestinal epithelial cell differentiation involves activation of p38 mitogen-activated protein kinase that regulates the homeobox transcription factor CDX2.

The intracellular signaling pathways responsible for cell cycle arrest and differentiation along the crypt-villus axis of the human small intestine remain largely unknown. p38 mitogen-activated protein kinases (MAPKs) have recently emerged as key modulators of various vertebrate cell differentiation processes. In order to elucidate further the mechanism(s) responsible for the loss of proliferative potential once committed intestinal cells begin to differentiate, the role and regulation of p38 MAPK with regard to differentiation were analyzed in both intact epithelium as well as in well established intestinal cell models recapitulating the crypt-villus axis in vitro. Results show that phosphorylated and active forms of p38 were detected primarily in the nuclei of differentiated villus cells. Inhibition of p38 MAPK signaling by 2-20 microm SB203580 did not affect E2F-dependent transcriptional activity in subconfluent Caco-2/15 or HIEC cells. p38 MAPK activity dramatically increased as soon as Caco-2/15 cells reached confluence, whereas addition of SB203580 during differentiation of Caco-2/15 cells strongly attenuated sucrase-isomaltase gene and protein expression as well as protein expression of villin and alkaline phosphatase. The binding of CDX2 to the sucrase-isomaltase promoter and its transcriptional activity were significantly reduced by SB203580. Pull-down glutathione S-transferase and immunoprecipitation experiments demonstrated a direct interaction of CDX3 with p38. Finally, p38-dependent phosphorylation of CDX3 was observed in differentiating Caco-2/15 cells. Taken together, our results indicate that p38 MAPK may be involved in the regulation of CDX2/3 function and intestinal cell differentiation.

Inhibition by deacetylase inhibitors of IL-1-dependent induction of haptoglobin involves CCAAT/Enhancer-binding protein isoforms in intestinal epithelial cells.

Intestinal epithelial cells participate in an acute phase response (APR) by responding to cytokines and by expressing acute phase protein genes. We hypothesized that butyrate, a fermentation product of the bacterial intestinal flora with deacetylase activity, affects the APR in intestinal epithelial cells. Sodium butyrate (NaBu) and Trichostatin A (TSA) induced alkaline phosphatase activity and histone H4 acetylation in IEC-6 rat intestinal epithelial cells treated with or without interleukin-1beta (IL-1). In contrast, both NaBu and TSA attenuated the IL-1-dependent induction of the acute phase protein gene haptoglobin, as well as C/EBPbeta and C/EBPdelta transcription factors mRNAs. Gel shift and supershift assays showed a strong decrease in the IL-1-induced C/EBPbeta and C/EBPdelta containing complexes binding to the HaptoA C/EBP DNA-binding site of the haptoglobin promoter, by NaBu and TSA. Furthermore, site-specific mutation of the HaptoA site abolished the NaBu- and TSA-dependent inhibition of haptoglobin, as determined by transient transfection assays. These results suggest that deacetylase inhibitors may regulate the IL-1 dependent induction of haptoglobin by down-regulating C/EBP isoforms, and that C/EBPs represent a target for the action of butyrate in the control of the APR of intestinal epithelial cells.

MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells.

The present report delineates the critical pathway in the G(1) phase involved in downregulation of p27(Kip1), a cyclin-dependent kinase inhibitor, which plays a pivotal role in controlling entry into the S phase of the cell cycle. In resting CCL39 fibroblasts and IEC-6 intestinal epithelial cells, protein levels of p27(Kip1) were elevated but dramatically decreased on serum stimulation, along with hyperphosphorylation of pRb and increased CDK2 activity. In both cell types, expression of ras resulted in an increase of basal and serum-stimulated E2F-dependent transcriptional activity and a reduction in p27(Kip1) protein levels as well. The role of the mitogen-activated protein (MAP) kinase cascade in p27(Kip1) reduction and S phase reentry was reinforced by the blockades of serum-induced E2F-dependent transcriptional activity and p27(Kip1) downregulation with the MKK-1/2 inhibitor PD-98059. In both cell lines, downregulation of p27(Kip1) was associated with a repression of its synthesis, an event mediated by the p42/p44 MAP kinase pathway. Using an antisense approach, we demonstrated that p27(Kip1) may control cell cycle exit in both cell types. These data indicate that activation of the MAP kinase cascade is required for S phase entry and p27(Kip1) downregulation in fibroblasts and epithelial cells.

Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108-15 cells.

In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108-15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108-15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108-15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108-15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 microM of the MEK inhibitor, PD98059, only moderately affected elongation, 50 microM PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108-15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.

Negative regulation of glucocorticoid-dependent induction of c-fos by ras in intestinal epithelial cells.

In order to investigate the regulatory mechanisms involved in the expression of fos and jun family members by glucocorticoids, and the effect of ras transformation in intestinal epithelial cells, we used the rat cell line IEC-6. Dexamethasone treatment induced transiently c-jun mRNAs, in contrast to the sustained expression of c-fos, whereas its effect on junB expression resulted in a later increase. Dexamethasone-dependent stimulation of c-fos and c-jun was modulated predominantly at the level of transcription. Sustained levels of induced c-fos and c-jun proteins were observed after dexamethasone treatment. AP-1 DNA-binding capacity of c-fos, and to a smaller extent c-jun, was increased by glucocorticoids later than after serum treatment. To analyse the effect of ras on the glucocorticoid response of AP-1 components, we studied several IEC-6 cell clones transformed by the Ha-ras oncogene. In comparison to normal cells, these transformants displayed increased AP-1 DNA-binding activity with higher levels of junB and variable levels of c-jun in the AP-1 complex. Ras transformation repressed the growth-inhibitory properties of glucocorticoids. Furthermore, ras inhibited the glucocorticoid-dependent induction of c-fos protein and mRNA, leading to changes in AP-1 composition as compared to normal cells. As assessed by transient transfection luciferase assays, glucocorticoids induced significantly a minimal promoter containing 3 copies of an AP-1 DNA-binding site as well as the murine c-fos -276 to +112 promoter in non-transformed cells. In contrast, glucocorticoid addition did not induce these constructs in two ras transformed cell lines. These results suggest that ras negatively modulates specific responses of intestinal epithelial cells to glucocorticoids.

Attenuation of haptoglobin gene expression by TGFbeta requires the MAP kinase pathway.

In addition to important roles in the regulation of cell growth and cell restitution, both pro- and anti-inflammatory effects have been ascribed to TGFbeta in intestinal epithelial cells. However, the mechanisms involved in TGFbeta-dependent anti-inflammatory activities remain to be determined. In the rat intestinal epithelial cell line IEC-6, TGFbeta attenuated the glucocorticoid-dependent increases in mRNA levels of the acute phase protein gene haptoglobin, and of C/EBP isoforms beta and delta. Supershift assays demonstrated a TGFbeta-mediated decrease in the binding of C/EBP isoforms beta and delta to the haptoA and haptoC C/EBP DNA-binding sites from the haptoglobin promoter. Mutations of both HaptoA and HaptoC sites abolished the glucocorticoid-dependent activation and the TGFbeta-mediated attenuation of the haptoglobin promoter, as assessed by transient transfection assays. TGFbeta induced p42/p44 MAP kinase activities. Treatment with the MEK 1/2 inhibitor PD 98059 abolished TGFbeta attenuation. These results suggest that C/EBP isoforms are involved both in the glucocorticoid-dependent induction and in the TGFbeta-mediated attenuation of haptoglobin expression. Furthermore, p42/p44 MAP kinases may function in a TGFbeta-dependent signaling pathway leading to attenuation of haptoglobin expression.

New hemodynamic approach to angiogenesis: color and pulsed Doppler ultrasonography.

RATIONALE AND OBJECTIVES: To determine the capacity of color and spectral Doppler ultrasonography (US) to quantify angiogenesis in vivo and to characterize low-resistance intratumor blood flow. METHODS: Thirty-two tumors, xenografted into mice, were studied with Doppler US. The number of intratumor vessels visualized with color Doppler US was compared with the density of microvessels and the number of vessels >100 microm determined by histologic examination. The resistance index and the peak systolic velocities were evaluated. RESULTS: The number of intratumor vessels visualized by color Doppler US was correlated with the number of vessels >100 microm (P<0.001) determined histologically. When vessel density was >30, intratumor vessels were always detected by color Dopper US. The resistance index and peak systolic velocities were significantly lower in intratumor than in peritumor vessels. CONCLUSIONS: Color Doppler US evaluated tumor angiogenesis accurately. Spectral analysis confirmed the low resistance of intratumor blood flow.

Activation of haptoglobin gene expression by cAMP involves CCAAT/enhancer-binding protein isoforms in intestinal epithelial cells.

CCAAT/enhancer-binding protein (C/EBP) isoforms are expressed in rodent intestine and in the rat intestinal epithelial cell line IEC-6 but their role remains to be determined. Treatment of IEC-6 cells with the adenylate cyclase activator forskolin led to coordinate induction of C/EBP isoforms alpha, beta and delta at the mRNA and protein levels. Transient transfection assays showed that their expression is controlled at the transcriptional level. Forskolin treatment induced haptoglobin mRNA levels. Electrophoretic mobility shift and supershift assays demonstrated an increase in DNA-binding activities of the three C/EBP isoforms to the haptoA and haptoC C/EBP DNA-binding sites of the proximal haptoglobin promoter. Site-specific mutations of both sites led to a decrease in transcriptional induction by forskolin, suggesting that C/EBP isoforms are involved in the cAMP-dependent regulation of the acute-phase protein gene haptoglobin in intestinal epithelial cells.

CCAAT/enhancer binding proteins beta and delta regulate alpha1-acid glycoprotein gene expression in rat intestinal epithelial cells.

Isoforms of CCAAT/enhancer binding protein (C/EBP) are expressed in rodent intestine as well as in the rat intestinal epithelial cell line IEC-6. However, no specific roles have been attributed to these isoforms in intestinal epithelial cells. To determine whether C/EBP family members could be implicated in the regulation of acute-phase response gene expression in intestinal epithelial cells, we have studied the effect of glucocorticoids on expression of the alpha1-acid glycoprotein gene and C/EBP isoforms in IEC-6 cells. Glucocorticoids induced alpha1-acid glycoprotein gene expression in these cells. This induction coincided with an increase of DNA-binding capacity of both C/EBPbeta and C/EBPdelta to the B1 and B2 C/EBP-interacting sites localized in the rat AGP promoter. Transforming growth factor beta, (TGFbeta), a cytokine involved in the transcriptional regulation of several acute-phase plasma proteins, antagonized the glucocorticoid-dependent induction of alpha1-acid glycoprotein gene expression. In parallel, TGFbeta downregulated the DNA-binding capacities of both the C/EBPbeta and C/EBPdelta isoforms. Mutations of the B1 or the B2 C/EBP-interacting site strongly reduced the responsiveness of the alpha1-acid glycoprotein promoter to glucocorticoids and TGFbeta. These results demonstrate a functional role for C/EBPbeta and C/EBPdelta in rat intestinal epithelial cells and suggest that these isoforms represent important modulators of the acute-phase response and of glucocorticoid, as well as TGFbeta, responsiveness.

Transcriptional regulation of pre-pro-endothelin-1 gene by glucocorticoids in vascular smooth muscle cells.

Endothelin is a potent vasoactive peptide involved in the maintenance of vascular tone and in pathophysiological states. Endothelin-1 synthesis is controlled at the transcriptional level. We report that glucocorticoids increase the pre-pro-endothelin-1 gene transcription rate in vascular smooth muscle cells. The effect of glucocorticoids is dose-dependent (EC50 approximately 2-3 nM) and completely blocked by co-incubation with the glucocorticoid antagonist RU 38486. The rise in pp-Et-1 steady state mRNA levels is rapid and transient with a maximal three-fold stimulation within one hour of glucocorticoid administration. Glucocorticoid treatment does not affect the half-life of pre-pro-endothelin-1 mRNA as shown by actinomycin D studies. Furthermore, cycloheximide treatment concomitantly with RU 28362 did not reverse the stimulatory effect of glucocorticoids on pre-pro-endothelin-1 mRNA levels. Nuclear run-on analysis shows that glucocorticoids increase the transcription rate of pre-pro-endothelin-1. Our results suggest a role for glucocorticoids in the regulation of biosynthesis and action of this important vasoactive peptide in vascular smooth muscle cells.

A role for p21ras in the angiotensin II AT2 receptor transduction pathway.

We have previously shown that the activation of the AT2 receptor of Ang II induced neurite outgrowth in NG108-15 cells. We also found that stimulation of NG108-15 cells with Ang II induced a rapid decrease in GTP-bound p21ras. In order to investigate the possible role of p21ras in Ang II-induced neuronal differentiation, we have established NG108-15 sublines which inducibly express a dominant inhibitory form of p21ras (p21N17Ras). We observed that IPTG-induced expression of p21N17Ras in these NG108-15 sublines induced the same morphological changes as does Ang II in control untransfected cells. Immunofluorescence labeling of beta-tubulin showed that expression of p21N17Ras induced neurite outgrowth and elongation. These observations were supported by Western blot analysis of the level of polymerized tubulin. These results strongly support the hypothesis that AT2 receptor-induced neuronal differentiation in NG108-15 cells is mediated by the inhibition of p21ras.

Transforming growth factor-beta1 is a potent inhibitor of glutathione synthesis in the lung epithelial cell line A549: transcriptional effect on the GSH rate-limiting enzyme gamma-glutamylcysteine synthetase.

Glutathione (GSH) is an essential antioxidant tripeptide that protects mammalian cells against oxidants and xenobiotics. Patients with fibrotic lung disorders have very low levels of GSH in their alveolar epithelial lining fluid (ELF), whereas transforming growth factor (TGF)-beta is overexpressed in their alveolar epithelial cells. We observed that TGF-beta1 increased susceptibility of the human alveolar epithelial cell line A549 to H2O2-mediated cytotoxicity (P < 0.05), decreased the activities of the antioxidant enzymes glutathione reductase and catalase by 31%, and markedly decreased GSH content in A549 cells (P < 0.01). GSH depletion was associated with an equivalent decrease in the activity of the rate-limiting enzyme in GSH synthesis, gamma-glutamylcysteine synthetase (gamma-GCS) (P < 0.01). Western blot analysis confirmed that the loss of gamma-GCS activity was associated with a marked decrease in gamma-GCS heavy subunit (gamma-GCShs) protein. TGF-beta1 suppressed the steady-state level of messenger RNA (mRNA) for the gamma-GCShs gene, with a maximal effect at 24 h. The half-life of gamma-GCShs mRNA was not affected by TGF-beta1, but transcription of the gene was downregulated as determined with nuclear run-on assays. Our findings indicate for the first time that TGF-beta1 is a potent inhibitor of GSH synthesis in human lung epithelial cells, and that the inhibition is mediated, at least in part, by a transcriptional effect on the gene encoding gamma-GCShs. Regulation of gamma-GCShs gene expression by TGF-beta1 is likely to play an important role in lower respiratory tract GSH homeostasis, and may represent a novel target for therapeutic efforts in lung fibrosis.