Biomedical subjects

Hendrik Ungefroren

Publications and source records attributed to Hendrik Ungefroren.

At least 19 recordsLinked to original sources

PubMed2007

Antitumor activity of ALK1 in pancreatic carcinoma cells.

In this study, the authors investigated the expression of activin receptor-like kinase 1 (ALK1) in pancreatic carcinoma and evaluated its potential role as a tumor suppressor in vitro and in vivo. Endogenous ALK1 expression was demonstrated by immunohistochemistry in both pancreatic tumor tissue and peritumoral normal tissue from 6 patients and by RT-PCR in 8/12 established pancreatic cancer cell lines. Ectopic expression of a constitutively active (ca) ALK1 mutant in TGF-beta sensitive PANC-1 and COLO-357 cells augmented transcriptional activation of a Smad2/3 responsive reporter, and slowed down basal growth in vitro. Both effects were further enhanced by TGF-beta/ALK5 stimulation, suggesting largely independent nuclear Smad signaling by both type I receptors. Upon orthotopic transplantation of PANC-1-caALK1 into immunodeficient mice, tumor size was strongly reduced and was associated with a lower microvessel density in the PANC-1-caALK1-derived tumors. In vitro, this mutant efficiently blocked TGF-beta-induced epithelial-to-mesenchymal transdifferentiation and suppressed TGF-beta/ALK5-mediated activation of the p38 MAPK pathway. Mechanistically, caALK1 silenced MyD118, an immediate TGF-beta target gene whose protein product, GADD45beta, couples Smad signaling to p38 activation. These results show that ALK1 activation in pancreatic tumor cells is antioncogenic by inducing ALK5-independent growth inhibition and by blocking TGF-beta/ALK5-mediated epithelial-to-mesenchymal transdifferentiation and, possibly, invasion and metastatic progression.

Activin Receptors, Type I↗

PubMed2006

Preoperative low-dose irradiation promotes long-term allograft acceptance and induces regulatory T cells in a porcine model of pulmonary transplantation.

BACKGROUND: A simplified conditioning protocol including single-dose preoperative thymic and low-dose whole body irradiation with or without subsequent donor bone marrow transplantation (BMTx) can be applied in porcine lung transplantation. We hypothesized that this protocol would prolong allograft survival. METHODS: Left-sided single lung transplantation from major histocompatibility complex (MHC)-mismatched donors was performed in 27 minipigs. Recipients received whole body (1.5 Gy) and thymic irradiation (7 Gy) before transplantation (IRR group, n=6), intravenous immunosuppression with methylprednisolone, cyclosporine, and azathioprine for 27 postoperative days (IS group, n=5) or both (IRR+IS group, n=10). BMTx group recipients were treated with irradiation, immunosuppression and a donor bone marrow infusion on postoperative day 1. Peripheral blood leukocyte phenotype and donor cell chimerism were monitored by flow cytometry. Purified CD25+ T cells from long-term survivors or rejecting animals were used for in vitro MLR suppression assays. RESULTS: Median graft survival was: IRR 12 days, IS 55 days, IRR+IS 239 days, and BMTx 80 days (P<0.0001). Early peripheral blood macrochimerism was substantial in both the IRR+IS and the BMTX group but was lost in all groups after day 80. The frequency and suppressive function of CD4+CD25+ T cells were enhanced in IRR+IS group long-term survivors. CONCLUSION: Although donor bone marrow infusion was not beneficial in our model, a substantial proportion of the animals treated with irradiation and a 28-day course of immunosuppression accepted their lung allografts long term. The mechanism involved in maintaining allograft tolerance may be based on peripheral T-cell regulation.

Animals↗

PubMed2006

WOFIE stimulates regulatory T cells: a 2-year follow-up of renal transplant recipients.

BACKGROUND: Initial interruption of immunosuppression for 72 hr was analyzed in renal transplant recipients according to Calne et al.'s "window of opportunity for immunologic engagement" (WOFIE) concept. METHODS: This pilot study was designed as a randomized, open-label, prospective trial of 40 recipients (20 in the WOFIE group, 20 in the control group) of cadaveric kidney transplants who were followed up for 2 years. Immunosuppression comprised tacrolimus (trough levels 5-8 ng/mL), daclizumab (1 mg per kilogram of body weight on day 0 and after 2, 4, 6, and 8 weeks), mycophenolate mofetil (1-2 g/day), and prednisolone (maintenance dose of 10 mg/day). After induction with daclizumab, prednisolone, and mycophenolate mofetil, immunosuppression was interrupted for 72 hr in the WOFIE group. Steroid withdrawal followed in both groups within 12 to 16 weeks posttransplant. RESULTS: Patient and graft survival did not differ significantly between the two cohorts. However, the WOFIE group experienced less acute rejection episodes and developed better graft function. Although all but one of the patients in the WOFIE group successfully discontinued steroid treatment, permanent steroid withdrawal was achieved in only 76.4% of the control group. After daclizumab discontinuation, the WOFIE group demonstrated an increase of CD4CD25 T cells in peripheral blood (P<0.05 vs. control group), which was stable over time and strongly correlated with a significantly higher expression level of Foxp3-mRNA. CONCLUSIONS: Initial interruption of immunosuppression for 72 hr correlates with the induction of regulatory immunologic mechanisms and allows early and reliable minimization of immunosuppressive treatment.

Adolescent↗

PubMed2006

Derivation, maintenance, and characterization of rat embryonic stem cells in vitro.

The in vitro differentiation of mouse embryonic stem (ES) cells into different somatic cell types such as neurons, endothelial cells, or myocytes is well established, and many mouse ES cell lines have been created so far. The establishment of rat ES cell lines, however, has proven to be difficult. Most attempts to culture rat ES cell lines and maintain them in an undifferentiated state have failed, so researchers were forced to abandon this system and use mouse ES cells. This chapter describes the long-term cultivation of an alkaline phosphatase-positive rat embryonic stem cell-like line (RESC) and their differentiation into neuronal, endothelial, and hepatic lineages. The RESCs can be characterized by typical growth in single cells as well as embryoid bodies when cultivated in the presence of leukemia inhibitory factor. RESC expressed stage-specific-embryonic antigen 1 and the major histocompatibility class 1 molecule. Neuronal differentiation is achieved by standard retinoic acid treatment and endothelial differentiation can be reproducibly induced by growth on or within Matrigel for 14 d. To induce expression of hepatocyte-specific antigens, RESCs were either grown in hepatocyte-conditioned media or in media containing different combinations of growth factors. The characterization of differentiated cells was done primarily by immunohistochemistry, enzyme-linked immunosorbent assay, and polymerase chain reaction.

Alkaline Phosphatase↗

PubMed2006

Stable expression of temperature-sensitive p53: a suitable model to study wild-type p53 function in pancreatic carcinoma cells.

Pancreatic adenocarcinoma is an extremely aggressive malignancy with a dismal prognosis. Inactivation of the p53 tumor-suppressor gene occurs in approximately 50% of primary tumors and is thought to account for a failure of the tumor cells to undergo growth arrest and apoptosis in response to chemotherapy. Hence, it is of interest to study the consequences of the restoration of wild-type (wt) p53 function in pancreatic carcinoma cells. Therefore, we retrovirally transduced temperature-sensitive (ts) human p53 into the p53-null pancreatic carcinoma cell line AsPC-1. ts p53 has a mutant phenotype at 37.5 degrees C, and a wt conformation at 32.5 degrees C. Stable expression of p53 in wt conformation caused upregulation of the p53 responsive gene p21Waf1/Cip1, and G1 growth arrest, but failed to induce Bax expression or apoptosis. In addition, we examined the effect of wt p53 expression on DNA damaging treatment. Interestingly, the doxorubicin- and radiation-induced S-/G2-phase arrests were suppressed by p53 in wt conformation. These results demonstrate that the ts p53/AsPC-1 model is suitable to investigate the effect of wt p53 restoration in pancreatic carcinoma cells.

Adenocarcinoma↗

PubMed2005

Catecholamine-synthesizing enzymes in the adult and prenatal human testis.

Catecholamines play functional roles in the mature and developing mammalian testis but the cell types responsible for their local synthesis are still controversially discussed. Here, we demonstrate that four enzymes involved in the biosynthesis of catecholamines, namely, tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DBH) and phenylethanolamine- N-methyltransferase (PNMT), are expressed in Leydig cells of the human testis. Tyrosine hydroxylase, the key enzyme of the biosynthesis of catecholamines, was localized to Leydig cells both at the transcript level (by RT-PCR analyses and by in situ hybridization assays) and at the protein level (by immunoblotting and by immunohistochemistry). The other enzymes were also demonstrated in Leydig cells by RT-PCR and immunohistochemical analyses. The presence of TH, AADC, DBH, and PNMT in human Leydig cells was found, in addition, by immunohistochemical approaches carried out on sections from prenatal human testes. Thus, the present study identifies the Leydig cells as the presumed sites of catecholamine production in both the mature and fetal human testes and further supports the previously recognized neuroendocrine characteristics of this cell type.

Aged↗

PubMed2005

Gemcitabine-mediated apoptosis is associated with increased CD95 surface expression but is not inhibited by DN-FADD in Colo357 pancreatic cancer cells.

This study investigates the role of caspase-8 and DN-FADD, an inhibitor of CD95-dependent caspase-8 activation, in gemcitabine-induced apoptosis of Colo357 pancreatic cancer cells. Gemcitabine-mediated apoptosis was monitored by the kinetics of caspase-8 activation and cytochrome c release. Gemcitabine treatment of Colo357 cells increased CD95 surface expression, raising the possibility of the involvement of CD95 in gemcitabine-mediated caspase-8 activation. However, ectopic expression of DN-FADD and treatment of cells with the antagonistic anti-CD95 antibody ZB4 both failed to suppress gemcitabine-induced apoptosis but substantially inhibited CD95-mediated apoptosis. DN-FADD, which surprisingly accumulated in nuclei of Colo357 cells, was unable to block caspase-8 activation mediated by either gemcitabine or CD95. These observations argue against a role of CD95 in gemcitabine-induced caspase-8 activation and reveal that the anti-apoptotic function of DN-FADD differs from caspase-8 inhibition in Colo357 cells.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Adhesion and Rac1-dependent regulation of biglycan gene expression by transforming growth factor-beta. Evidence for oxidative signaling through NADPH oxidase.

Both transforming growth factor-beta (TGF-beta)-induced expression of biglycan (BGN) and activation of p38 MAPK have been implicated in cellular adhesion and migration. Here, we analyzed the role of adhesive events and the small GTPase Rac1 in TGF-beta regulation of BGN. TGF-beta1 induction of BGN expression and activation of p38 was abolished or strongly reduced when cells were kept in suspension or exposed to either the actin cytoskeleton-disrupting agent cytochalasin D or a specific chemical Rac1 inhibitor. Ectopic expression of a dominant negative mutant (T17N) of Rac1 abrogated both TGF-beta-induced p38 MAPK activation and BGN up-regulation but did not affect TGF-beta-induced phosphorylation of Smad3 or transcriptional induction of Growth Arrest DNA Damage 45beta, previously shown to be crucial for TGF-beta regulation of BGN. Overexpression of wild type Rac1 greatly enhanced the TGF-beta effect on BGN in adherent cells, whereas ectopic expression of constitutively active Rac1 (Q61L) activated p38 and in the presence of exogenous TGF-beta was able to rescue BGN expression in nonadherent cells. Endogenous Rac1 was activated by TGF-beta treatment in PANC-1 cells in an adhesion-dependent fashion. Like Rac1-T17N, the NADPH oxidase inhibitor diphenylene iodonium and the tyrosine kinase inhibitor herbimycin A blocked TGF-beta-induced p38 activation and BGN expression, suggesting that Rac1 exerts its effect on BGN and p38 through increasing NADPH oxidase activity and subsequent production of reactive oxygen species. These results show that the TGF-beta effect on BGN is dependent on cell adhesion and that activated Rac1, presumably acting through NADPH oxidase(s), is necessary but not sufficient for TGF-beta-induced BGN expression.

Biglycan↗

PubMed2005

Human monocyte-derived neohepatocytes: a promising alternative to primary human hepatocytes for autologous cell therapy.

BACKGROUND: There is growing interest in new therapeutic options for the treatment of end-stage liver diseases. In addition to mechanical devices supporting liver function, such as bioreactors, the transplantation of hepatocyte-like cells derived from (adult) stem cells offer great perspectives. We have generated hepatocyte-like (NeoHep) cells from terminally differentiated peripheral blood monocytes and, in this study, have evaluated these cells as a possible tool for autologous cell therapy. METHODS: Peripheral blood monocytes were cultured under conditions that promote hepatocyte-like differentiation and were characterized for hepatocyte marker expression by reverse-transcriptase polymerase chain reaction, immunohistochemistry, and immunoblotting and for specific secretory and metabolic functions with the appropriate biochemical assays. RESULTS: NeoHep cells resembled primary human hepatocytes with respect to morphology, expression of hepatocyte markers (albumin, cytochrome P450 isoenzymes, asialoglycoprotein receptor, coagulation factor VII), various secretory and metabolic functions (albumin secretion, urea production, lactate formation, and lactate dehydrogenase and aspartate transaminase release), and drug detoxification activities (phase I metabolization of ethoxycoumarin into 7OH-coumarin after stimulation with 3-methylcholanthren, induction of CYP3A4 activity, and phase II metabolization through UDP-glucuronidation of 4-methyl-umbelliferone). CONCLUSIONS: These data convincingly show that NeoHep cells display a phenotype and specific in vitro metabolic functions that are quantitatively and qualitatively comparable in part with those of primary human hepatocytes. These cells could thus be clinically applied in an autologous setting for the treatment of end-stage liver diseases or for improving liver function in patients who have undergone critical liver-mass resection.

Biomarkers↗

PubMed2005

Differentiation of in vitro-modified human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells.

BACKGROUND & AIMS: Adult stem cells provide a promising alternative for the treatment of diabetes mellitus and end-stage liver diseases. We evaluated the differentiation potential of human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells. METHODS: Monocytes were treated with macrophage colony-stimulating factor and interleukin 3 for 6 days, followed by incubation with hepatocyte and pancreatic islet-specific differentiation media. Cells were characterized by flow cytometry, gene-expression analysis, metabolic assays, and transplantation for their state of differentiation and tissue-specific functions. RESULTS: In response to macrophage colony-stimulating factor and interleukin 3, monocytes resumed cell division in a CD115-dependent fashion, which was associated with a down-regulation of the PRDM1 and ICSBP genes. These programmable cells of monocytic origin were capable of differentiating into neohepatocytes, which closely resemble primary human hepatocytes with respect to morphology, expression of hepatocyte markers, and specific metabolic functions. After transplantation into the liver of severe combined immunodeficiency disease/nonobese diabetic mice, neohepatocytes integrated well into the liver tissue and showed a morphology and albumin expression similar to that of primary human hepatocytes transplanted under identical conditions. Programmable cells of monocytic origin-derived pancreatic neoislets expressed beta cell-specific transcription factors, secreted insulin and C peptide in a glucose-dependent manner, and normalized blood glucose levels when xenotransplanted into immunocompetent, streptozotocin-treated diabetic mice. Programmable cells of monocytic origin retained monocytic characteristics, notably CD14 expression, a monocyte-specific methylation pattern of the CD115 gene, and expression of the transcription factor PU.1. CONCLUSIONS: The ability to reprogram, expand, and differentiate peripheral blood monocytes in large quantities opens the real possibility of the clinical application of programmable cells of monocytic origin in tissue repair and organ regeneration.

Albumins↗

PubMed2005

Generation of human hepatocytes by stem cell technology: definition of the hepatocyte.

Since 1999, numerous articles have reported the generation of hepatocytes from different types of extrahepatic stem or precursor cells. This opens exciting new possibilities for pharmacology and toxicology, as well as for cell therapy. Hepatocyte marker expression, including albumin, cytokeratin 18, c-met, alpha-fetoprotein and cytochrome P450 3A4 and -2B6, has been observed after transplantation of different types of human stem cells into the liver of laboratory animals or in vitro after incubation with cytokines. These intriguing observations have prompted scientists to classify stem cell-derived cell populations as hepatocytes. However, this conclusion may be premature. It has been shown that factors of the liver microenvironment can induce expression of a limited number of hepatocyte marker genes in nonhepatic cell types. To conclude on the grounds of a limited number of markers that these cells are true hepatocytes is not indicated. In this case one should carefully evaluate crucial hepatocyte-defining enzymatic properties. The present article: i) reviews studies describing the fate of extrahepatic human stem and precursor cells in livers of laboratory animals, including the possibility of cell fusion; and ii) critically discusses the phenotype of stem cells after application of various differentiation protocols aimed at generating human hepatocytes. In addition, the necessary criteria needed for defining a true hepatocyte are suggested. Establishing the necessary properties for stem cell-derived hepatocytes is timely and reasonable, and thus avoids further misleading semantic confusion. Finally, it is essential to understand that the definition of a bona fide hepatocyte should not be limited to qualitative assays, such as reverse transcriptase polymerase chain reaction and immunohistochemistry, but has to include a quantitative analysis of enzymatic activities, which allows direct comparison with primary hepatocytes. Although the stem cell-derived-hepatocyte does not yet exist there is a good chance that this aim may be achieved in the future.

Animals↗

PubMed2004

Transforming growth factor-beta (TGF-beta) type I receptor/ALK5-dependent activation of the GADD45beta gene mediates the induction of biglycan expression by TGF-beta.

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.

Activin Receptors, Type I↗

PubMed2004

Resistance of pancreatic cancer to gemcitabine treatment is dependent on mitochondria-mediated apoptosis.

Palliative chemotherapy with gemcitabine, a common mode of treatment of pancreatic cancer, has little influence on patients' survival. We investigated the impact of anti-apoptotic Bcl-xL protein and its antagonist Bax on gemcitabine-induced apoptosis in human pancreatic carcinoma cells in vitro and in vivo. The level of Bcl-xL and Bax expression was determined in 3 established pancreatic cancer cell lines that differ in their sensitivity to gemcitabine-mediated apoptosis. Bcl-xL and Bax genes were transduced into Colo357 cells by retroviral infection. In addition, cells were transfected with c-FLIP to assess involvement of CD95 and caspase-8. The impact of Bax/Bcl-xL expression on gemcitabine-sensitivity in vivo was evaluated in orthotopic Colo357 tumors in SCID mice. The apoptotic index revealed a strong inverse correlation between Bcl-xL expression and gemcitabine-induced apoptosis in the pancreatic carcinoma cell lines tested. Caspase-8 and Bid were cleaved in Colo357 cells exposed to gemcitabine, and there was no correlation with either Bcl-xL or with Bax expression. In contrast, the lack of mitochondrial transmembrane potential transition, release of cytochrome-c and absence of caspase-9- and PARP-cleavage showed a strong correlation with Bcl-xL expression. Expression of c-FLIP significantly increased the resistance towards gemcitabine. Orthotopically growing Colo357-bcl-xl tumors in SCID mice were refractory to gemcitabine treatment, and in contrast to the in vitro data, Colo357-bax tumors exhibited a 12-fold greater tumor regression than Colo357-wild-type tumors in the control group. Gemcitabine-induced apoptosis involves the mitochondria-mediated signaling pathway. A functional restoration of this pathway appears to be essential to overcome the resistance mechanisms of pancreatic tumor cells and to improve the response to therapy as demonstrated by Bax overexpression in a clinically relevant tumor model.

Animals↗

PubMed2004

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity.

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.

Animals↗

PubMed2004

Stem cell-mediated tolerance inducing strategies in organ transplantation.

The scope of possible tools to modulate the recipients immune response towards tolerance induction basically includes deletional and non deletional mechanisms, which are currently targeted by various strategies including monoclonal antibodies, cytokine deviation, chimerism induction and the support of regulating T-cells. Here we summarize the main findings in the field derived from experimental animal studies and currently performed clinical studies. This review focuses to give a clinically relevant overview over relevant tolerance inducing concepts, taking into consideration risk profiles and clinical efficacy associated with specific immunosuppressive regiments currently applied in the clinical setting of transplantation.

Animals↗

PubMed2003

PKCmu prevents CD95-mediated apoptosis and enhances proliferation in pancreatic tumour cells.

Loss of growth control and a marked resistance to apoptosis are considered major mechanisms driving tumour progression. Protein kinases C (PKC) have been shown to be important in the regulation of proliferation and apoptosis. In this report, we investigated the role of the PKC-like kinase PKCmu in the control of these processes in pancreatic adenocarcinoma cells. We demonstrate that in these cells, PKCmu expression strongly correlates with resistance to CD95-induced apoptosis. Inhibition of PKCmu with Goe6983 sensitized resistant cells to CD95-induced apoptosis. In CD95-sensitive Colo357 cells, forced overexpression of PKCmu strongly reduced CD95-mediated apoptosis, an effect that could be reversed by pretreatment with Goe6983. In addition, PKCmu overexpression led to a strongly enhanced cell growth and to a significant increase of telomerase activity. In an attempt to identify the signalling pathways affected by PKCmu, we identified the antiapoptotic proteins c-FLIPL and survivin to be strongly upregulated in PKCmu overexpressing cells. Immunohistochemical analysis of pancreatic tumour tissue of 48 patients and 10 normal pancreatic tissues revealed marked overexpression of PKCmu in tumours. In conclusion, we showed that PKCmu controls proliferative, as well as anti-apoptotic, signalling pathways and therefore plays an important role in acquiring the malignant phenotype of pancreatic tumours.

Adenocarcinoma↗

PubMed2003

Angiotensin II AT(1)-receptor induces biglycan in neonatal cardiac fibroblasts via autocrine release of TGFbeta in vitro.

OBJECTIVE: After myocardial infarction, angiotensin II (AngII) promotes ventricular remodeling and deposition of extracellular matrix (ECM), e.g., collagen type 1 and 3. Whether AngII regulates the expression of small leucine-rich proteoglycans (SLRP) which are important modulators of collagen fibrillogenesis and are induced after experimental myocardial infarction in rats is not known. The aim of the present study was therefore to analyse in cultured cardiac fibroblasts the expression and secretion of the SLRP biglycan in response to AngII. METHODS: Cardiac fibroblasts were isolated from neonatal Wistar Kyoto rats and used in the first passage. Expression of AT(1)- and AT(2)-receptors was verified by RT-PCR. Expression of protoeglycans was analyzed after metabolic labeling with [35S]-sulfate, by SDS-PAGE and Western analysis. In addition, mRNA expression was examined by means of RT-PCR and Northern analysis. The activity of the biglycan promoter was analyzed using three biglycan promoter-luciferase fusion constructs. RESULTS: Biglycan was found to be the predominant proteoglycan produced by neonatal cardiac fibroblasts in vitro. In response to AngII (10(-7) M), secretion of total [35S]-labeled proteoglycans and mRNA of biglycan were increased to 116+/-1.8% and 121+/-11% (n=5, mean+/-S.E.M.) of unstimulated controls, respectively. Biglycan induction in response to AngII was sensitive to Losartan (10(-5) M) and unaffected by PD123177 (10(-6) M), suggesting that the AT(1)-receptor mediates the induction of biglycan. Direct activation of the biglycan promoter downstream of the AT(1)-receptor was excluded by promoter activity assays. Instead, increased release of transforming growth factor beta 1 (TGFbeta1) was detected by ELISA in response to AT(1)-receptor stimulation. Furthermore, neutralising antibodies to TGFbeta1 inhibited biglycan induction in response to AngII. CONCLUSION: The results indicate that in cardiac fibroblasts AngII via the AT(1)-receptor causes autocrine release of TGFbeta1, which in turn induces biglycan expression and secretion.

Angiotensin II↗

PubMed2003

Regulation of biglycan gene expression by transforming growth factor-beta requires MKK6-p38 mitogen-activated protein Kinase signaling downstream of Smad signaling.

Several signaling pathways have been implicated in mediating TGF-beta1-induced extracellular matrix production and fibrosis. We have shown recently that induction of biglycan (BGN) expression by TGF-beta1 depended on a functional Smad pathway (Chen, W.-B., Lenschow, W., Tiede, K., Fischer, J. W., Kalthoff, H., and Ungefroren, H. (2002) J. Biol. Chem. 277, 36118-36128). Here, we present evidence that the ability of TGF-beta 1 to induce BGN mRNA, in addition to Smads, requires p38 MAPK signaling, because 1) pharmacological inhibitors of p38 dose-dependently inhibited the TGF-beta effect without significantly affecting the transcriptional activity of a constitutively active mutant of the TGF-beta type I receptor or Smad2 phosphorylation at concentrations up to 10 microm, 2) the up-regulation of BGN mRNA was preceded by a delayed increase in the phosphorylation of p38 and its upstream activator MKK6 in TGF-beta 1-treated PANC-1 cells, 3) inhibition of the p38 pathway by stable retroviral transduction with a dominant negative mutant of either p38 or MKK6 reduced TGF-beta 1-induced BGN mRNA expression, and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented the TGF-beta 1 effect on BGN mRNA. We further demonstrate that the (delayed) p38 activation by TGF-beta 1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-beta-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-beta1 induction of p38 activation and, as shown earlier, BGN mRNA expression; finally, re-expression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-beta-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation. These results clearly show that TGF-beta induction of BGN expression in pancreatic cells requires activation of MKK6-p38 MAPK signaling downstream of Smad signaling and provide a mechanistic clue to the up-regulation of BGN seen in inflammatory response-related fibrosis and desmoplasia.

Base Sequence↗