Characterization of β-D-xyloside-initiated glycosaminoglycan synthesized by human skin fibroblasts in the presence of tunicamycin

Human skin fibroblasts were incubated with a fluorogenic xyloside, 4-methylumbelliferyl--D-xyloside (Xyl-MU), in the presence or absence of tunicamycin. The xyloside-initiated glycosaminoglycans (GAG-MUs) were isolated from the culture medium, and their structures characterized. When the cells were incubated with Xyl-MU in the presence of 0.2 g ml–1 tunicamycin, the synthesis of GAG-MU was increased about three fold, compared with the control value in the absence of tunicamycin (cells exposed to Xyl-MU alone). The structures of GAG-MUs synthesized in the presence or absence of tunicamycin were compared by HPLC analysis using gel-filtration and ion-exchange columns, enzymatic digestion, and unsaturated disaccharide composition analysis. The data indicated that cells incubated with tunicamycin produced more undersulfated and shorter GAG-MUs than cells without tynicamycin. These results suggest that tunicamycin inhibits the elongation and sulfation of glycosaminoglycan (GAG) chains and that, as a result, GAG-MUs with shorter chains and undersulfated residues, but possessing a large number of GAG chains, are synthesized in the presence of tunicamycin.     

Interconversion of estrone and estradiol-17β in lung slices of the adult human

The metabolism of tritium-labeled estrone and estradiol-17β in slices of lung tissue obtained from an adult human was studied: estrone was identified as the only metabolite of estradiol-17β and estradiol-17β as the exclusive product of estrone metabolism. Product formation remained linear as a function of time of incubation up to 3 h and of wet lung tissue mass up to 300 mg/ml. At equimolar substrate concentrations, the rates of estrone formation were at least 2-fold greater than those of estradiol-17β. The apparent K_M of 17β-hydroxysteroid oxidoreductase for estrone was 11 μM and that for estradiol-17β was 10 μM. These results are suggestive that the human lung enzyme binds estrone and estradiol-17β with similar affinities; however, the oxidative pathway is favored as indicated by the greater V_max attained in the formation of estrone. It is possible that, in vivo, the human lung constitutes a site for estradiol-17β inactivation to estrone as well as a site for the conversion of estrone to estradiol-17β. This last process may become particularly important in instances in which the ovaries have ceased to function and secrete estradiol-17β, e.g. the postmenopausal women.     

Repeated exposure of human fibroblasts to ionizing radiation reveals an adaptive response that is not mediated by interleukin-6 or TGF-β

Exposing cells to a low dose can protect them against a subsequent higher exposure. This phenomenon is known as adaptive response and is frequently observed in a variety of cells. Even though similarities are suspected with other non-targeted effects, such as bystander effects, the exact mechanism behind adaptive response is not fully clarified. In this study human primary fibroblasts were tested for their response to ionizing radiation (IR) after administrating a low priming dose (0.1-0.5Gy). Both the abundance of γH2AX as a marker for double-stranded breaks and the levels of cytokines, secreted in the medium, were monitored in time. Upon challenge, IR-primed cells showed modified γH2AX spot size distributions and altered repair kinetics, consistent with an adaptive response. In addition, 24h after priming with IR, four cytokines were significantly upregulated in the medium - GM-CSF (1.33×); IL6 (4.24×); IL8 (1.33×); TGF-β (1.46×). In order to mimick the protective effect of IR priming, we primed the cells with either IL6 or TGF-β. This did not elicit an altered γH2AX response as observed in IR-primed cells, indicating that the adaptive response in these primary fibroblasts is regulated in an IL-6 and TGF-β independent manner.     

Regulation of TGF-β storage and activation in the human idiopathic pulmonary fibrosis lung

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-β (TGF-β) activation. Although TGF-β activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-β storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-β. Latent TGF-β-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-β signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-β signaling activity, respectively, suggesting that LTBP-1-mediated TGF-β activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-β-activating integrin β8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-β storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-β availability and activation in different pulmonary compartments in the fibrotic lung.     

N-Acetylglucosaminyltransferase V regulates TGF-β response in hepatic stellate cells and the progression of steatohepatitis

N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in tumor metastasis and carcinogenesis. Although the expression of GnT-V is induced in chronic liver diseases, the biological meaning of GnT-V in the diseases remains unknown. The aim of this study was to investigate the effects of GnT-V on the progression of chronic hepatitis, using GnT-V transgenic (Tg) mice fed a high fat and high cholesterol (HFHC) diet, an experimental model of murine steatohepatitis. Although enhanced hepatic lymphocytes infiltration and fibrosis were observed in wild-type (WT) mice fed the HFHC diet, they were dramatically prevented in Tg mice. In addition, the gene expression of inflammatory Th1 cytokines in the liver was significantly decreased in Tg mice than WT mice. Inhibition of liver fibrosis was due to the dysfunction of hepatic stellate cells (HSCs), which play pivotal roles in liver fibrosis through the production of transforming growth factor (TGF)-β1. Although TGF-β1 signaling was enhanced in Tg mouse-derived HSCs (Tg-HSCs) compared with WT mouse-derived HSCs (WT-HSCs), collagen expression was significantly reduced in Tg-HSCs. As a result from DNA microarray, cyclooxygenase-2 (COX2) expression, known as a negative feedback signal for TGF-β1, was significantly elevated in Tg-HSCs compared with WT-HSCs. Prostaglandin E2 (PGE2), the product of COX2, production was also significantly elevated in Tg-HSCs. COX2 inhibition by celecoxib decreased PGE2 and increased collagen expression in Tg-HSCs. In conclusion, GnT-V prevented steatohepatitis progression through modulating lymphocyte and HSC functions.     

Production of albumin and α-fetoprotein in primary culture of fetal human liver cells on collagenous substrata in the presence of hydrocortisone

Summary When primary cultures of fetal human liver cells established on type I collagen gels were compared to sister cultures developed on tissue culture plastic, the cells in contact with type I collagen secreted albumin at a higher rate than those without contact. The albumin secretion was dependent on the presence of hydrocortisone (HC) in the medium. Also, α-fetoprotein (AFP), of which the level decreased gradually and became undetectable after 6 d regardless of the presence or absence of HC in the cells cultured on plastic, was maintained for longer periods of time by plating the cells on type I collagen gels in the presence of HC. Different secretion rates of albumin and AFP were observed after Day 13 and Day 16, respectively, between cells maintained on type I collagen gels and those on film plastic. The cells secreted larger amounts of both albumin and AFP in plates coated with type IV or I collagens than with fibronectin after Day 10. The cells cultured on type I collagen gels were cuboidal in shape, whereas those on plastic were flattened in cultures with HC. These data indicate that the secretion of human albumin and AFP is facilitated by synergies between HC and collagenous substrata.     

Overexpression of MMP13 in human osteoarthritic cartilage is associated with the SMAD-independent TGF-β signalling pathway

IntroductionIn vitro and animal model of osteoarthritis (OA) studies suggest that TGF-β signalling is involved in OA, but human data is limited. We undertook this study to elucidate the role of TGF-β signalling pathway in OA by comparing the expression levels of TGFB1 and BMP2 as ligands, SMAD3 as an intracellular mediator, and MMP13 as a targeted gene between human osteoarthritic and healthy cartilage.MethodsHuman cartilage samples were collected from patients undergoing total hip/knee joint replacement surgery due to primary OA or hip fractures as controls. RNA was extracted from the cartilage tissues. Real-time quantitative PCR was performed to measure gene expression. Mann-Whitney test was utilized to compare the expression levels of TGFB1, BMP2, SMAD3 and MMP13 in human cartilage between OA cases and controls. Spearman’s rank correlation coefficient (rho) was calculated to examine the correlation between the expression levels of the four genes studied and non-parametric regression was used to adjust for covariates.ResultsA total of 32 OA cases (25 hip OA and 7 knee OA) and 21 healthy controls were included. The expression of TGFB1, SMAD3, and MMP13 were on average 70 %, 46 %, and 355 % higher, respectively, whereas the expression of BMP2 was 88 % lower, in OA-affected cartilage than that of controls (all p < 0.03), but no difference was observed between hip and knee OA (all p > 0.4). The expression of TGFB1 was correlated with the expression of SMAD3 (rho = 0.50, p = 0.003) and MMP13 (rho = 0.46, p = 0.007) in OA-affected cartilage and the significance became stronger after adjustment for age, sex, and BMI. The expression of BMP2 was negatively correlated with both TGFB1 (rho = −0.50, p = 0.02) and MMP13 (rho = −0.48, p = 0.02) in healthy cartilage, but the significance was altered after adjustment for the covariates. There was no correlation between the expression of SMAD3 and MMP13.ConclusionsOur results demonstrate that MMP13 expression is associated with an increased expression of TGFB1 in OA-affected cartilage, possibly through SMAD-independent TGF-β pathway. Furthermore, TGF-β/SMAD3 is overactivated in OA cartilage; yet, the consequence of this overactivation remains to be established.     

Focal adhesion kinase determines the fate of death or survival of cells in response to TNFα in the presence of actinomycin D

We speculated that focal adhesion kinase (FAK) might play a critical role in the TNFα-induced cell death. In this study, we found that FAK−/− cells are more sensitive to TNFα-induced apoptosis in the presence of actinomycin D (Act D) compared to FAK+/− cells. Prosurvival pathways are activated by the rapid recruitment of complex I, comprising TNFR1, TRADD, RIP and TRAF2, which leads to the activation of the NF-κB pathway. On the other hand, proapoptotic pathways are activated by complex II, the death-inducing signaling complex (DISC), which contains TNFR1, TRADD, RIP, and FADD, and procaspase-8 proteins. As TNFR1, TRADD, and RIP are included in both Complex I and DISC, we speculated that RIP might be a key protein. Coimmunoprecipitation assays revealed that RIP is included in complex I in FAK+/− cells, and FAK was associated with RIP. On the other hand, RIP is included in DISC in FAK−/− cells. FAK might be a key protein in the formation of complex I and the activation of NF-κB. Furthermore, Akt was activated in FAK+/− cells, but not FAK−/− cells. In conclusion, we first demonstrated that FAK determines the pathway leading to death or survival in TNFα/ActD-stimulated fibroblasts.     

TGF-β-mediated downregulation of microRNA-196a contributes to the constitutive upregulated type I collagen expression in scleroderma dermal fibroblasts

Previous reports indicated the significance of the TGF-β signaling in the pathogenesis of systemic sclerosis. We tried to evaluate the possibility that microRNAs (miRNAs) play a part in the type I collagen upregulation seen in normal fibroblasts stimulated with exogenous TGF-β and systemic sclerosis (SSc) fibroblasts. miRNA expression profile was evaluated by miRNA PCR array and real-time PCR. The protein expression of type I collagen was determined by immunoblotting. In vivo detection of miRNA in paraffin section was performed by in situ hybridization. Several miRNAs were found to be downregulated in both TGF-β-stimulated normal fibroblasts and SSc fibroblasts compared with normal fibroblasts by PCR array. Among them, miR-196a expression was decreased in SSc both in vivo and in vitro by real-time PCR or in situ hybridization. In SSc fibroblasts, miR-196a expression was normalized by TGF-β small interfering RNA. miR-196a inhibitor leads to the overexpression of type I collagen in normal fibroblasts, whereas overexpression of the miRNA resulted in the downregulation of type I collagen in SSc fibroblasts. In addition, miR-196a was detectable and quantitative in the serum of SSc patients. Patients with lower serum miR-196a levels had significantly higher ratio of diffuse cutaneous SSc:limited cutaneous SSc, higher modified Rodnan total skin thickness score, and higher prevalence of pitting scars than those without. miR-196a may play some roles in the pathogenesis of SSc. Investigation of the regulatory mechanisms of type I collagen expression by miR-196a may lead to new treatments using miRNA.     

Does the response to cocaine differ as a function of sex or hormonal status in human and non-human primates?

Stimulant abuse continues to be a growing problem among women. Over the last 10-15 years, an increasing number of studies have focused on factors that may be implicated in stimulant abuse in women as compared to men, including the role of hormonal fluctuations across the menstrual cycle. Numerous preclinical studies have documented that female rodents are more sensitive than male rodents to the behavioral effects of stimulant administration and the hormone estradiol is involved in the enhanced response to stimulants observed in females. In contrast, fewer studies have been conducted in humans and non-human primates addressing the role of sex and gonadal hormones on the effects of cocaine. This review paper presents a recent update on data collected in our Human Cocaine Challenge Laboratory and our Non-human Primate Laboratory, including analysis of cocaine pharmacokinetics, sex differences, the menstrual cycle, and the role of progesterone in modulating the response to cocaine. Our studies indicate that there is minimal evidence that the response to intranasal cocaine varies across the menstrual cycle or between men and women. In contrast, the response to smoked cocaine is greater in the follicular phase than the luteal phase and differences between men and women generally only emerge when men are compared to women in the luteal phase. In terms of potential hormonal mechanisms for these differences, the hormone progesterone attenuates the subjective response to cocaine. With respect to cocaine self-administration, there are minimal changes across the menstrual cycle in both humans and non-human primates. Thus, there is converging evidence across a range of species that the behavioral effects of cocaine (1) differ between males and females, (2) differ in relation to hormonal fluctuations, (3) can be attenuated by progesterone (at least in females), and (4) do not appear to be related to differences in cocaine pharmacokinetics.     

Fetal concentrations of the growth factors TGF-α and TGF-β1 in relation to normal and restricted fetal growth at term

Transforming growth factor-α (TGF-α) and TGF-β1 are major anti-inflammatory cytokines and substantially contribute to normal pregnancy outcome. TGF-α stimulates placental mitosis, whereas TGF-β1 is a critical regulator of trophoblast invasion and fetal growth. We aimed to study cord blood TGF-α and TGF-β1 concentrations in intrauterine-growth-restricted (IUGR, usually associated with abnormal trophoblast invasion, uteroplacental vascular insufficiency and enhanced inflammation) and appropriate-for-gestational-age-(AGA) pregnancies, and investigate possible correlations of the above concentrations with several demographic parameters of infants at birth. Plasma TGF-α and TGF-β1 concentrations were determined by ELISA in 154 mixed arterio-venous cord blood samples from IUGR (n=50) and AGA (n=104) singleton full-term infants. After controlling for possible confounding factors (gender, birth-weight, gestational age, maternal age and parity), cord blood TGF-α and TGF-β1 concentrations were significantly higher in IUGR than AGA group (b=0.402, SE=0.179, p=0.027 and b=0.152, SE=0.061, p=0.014, respectively). Delivery mode had an effect on cord blood TGF-α and TGF-β1 concentrations, both being elevated in cases of vaginal delivery (b=-0.282, SE=0.117, p=0.018 and b=-0.123, SE=0.059, p=0.038, respectively). In conclusion, higher cord blood TGF-α and TGF-β1 concentrations may represent a compensatory response to the inflammatory process characterizing the IUGR state. Additionally, higher cord blood TGF-β1 concentrations in IUGRs could be attributed to increased shear stress, resulting from abnormal blood flow in IUGR fetal blood vessels. Finally, vaginal delivery-associated cytokine release may account for elevated TGF-α and TGF-β1 concentrations.     

Sex specific response of cultured human bone cells to ERα and ERβ specific agonists by modulation of cell proliferation and creatine kinase specific activity

We have previously reported that human cultured bone cells (hObs) respond to estradiol-17β (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERβ specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERβ mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERβ pathways in the effects of estrogens on hObs, with a yet unknown mechanism.     

The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma

Background

Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown.

Methodology/Principal Findings

We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student''s t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells.

Conclusion

An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.