Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.     

Increased TGFbeta type II receptor expression suppresses the malignant phenotype and induces differentiation of human neuroblastoma cells

TGFbeta can modulate neuroblastoma (NB) cell proliferation and differentiation in vitro. In this study we used a NB cell line (LAN-5) which has been shown to partially respond to TGFbeta and to present high levels of TGFbeta receptor type I and low levels of receptor type II (TbetaRII) on the cell surface. To evaluate the role of TbetaRII in mediating TGFbeta effects, LAN-5 cells were transfected with an expression vector containing the human full-length TbetaRII cDNA or with the empty vector pcDNA3. Compared to control CLV3 cells (transfected with empty plasmid) and parental LAN-5 cells, isolated neomycin-resistant clones (CL1 and CL3) expressed higher levels of TbetaRII, had reduced cell growth rate in vitro, and were unable to form tumors in vivo. Furthermore, isolated clones modified their morphology, assuming a terminally differentiated neuronal phenotype. Immunocytochemical staining demonstrated a basal increased expression of neural-specific markers, such as axonal growth-associated protein (GAP43) and neurofilaments (NF200). TGFbeta treatment further increased the synthesis of NF200 and GAP43 in the transfected clones as revealed by Western blot analysis. These data indicate that TbetaRII overexpression potentiates the TGFbeta signal transduction pathway, reverting NB cell neoplastic phenotype with the reduction of proliferation rate and the induction of terminal maturation.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

Diabetes and tumor formation in transgenic mice expressing Reg I

To examine the effect of overexpressed regenerating gene (Reg) I on pancreatic beta-cells, we generated transgenic mice expressing Reg I in islets (Reg-Tg mice). Three lines of Reg-Tg mice were established. In line-1 Reg-Tg mice, the expression level of Reg I mRNA in islets was 7 times higher than those in lines 2 and 3 of Reg-Tg mice, and line 1 mice developed diabetes by apoptosis of beta-cells, as well as various malignant tumors. In addition to the decrease in beta-cells, compensatory islet regeneration and proliferation of ductal epithelial cells were observed in line-1 Reg-Tg mice. Because Reg I protein was secreted primarily into pancreatic ducts from acinar cells, it may primarily stimulate the proliferation of ductal epithelial cells, and not beta-cells, and their differentiation into islets. Moreover, the tumor-promoting activity of Reg I protein should be considered for its possible clinical applications.     

Immunohistochemical expression of FGF-2, PDGF-A, VEGF and TGF beta RII in the pancreas in the course of ischemia/reperfusion-induced acute pancreatitis.

Acute pancreatitis leads to pancreatic damage followed by subsequent regeneration. The aim of our study was to evaluate the presence of growth factors in the course of spontaneous pancreatic regeneration after ischemia/reperfusion (I/R)-induced pancreatitis. METHODS: In rats, I/R was evoked by clamping of splenic artery for 30 min followed by reperfusion. Rats were sacrificed 1, 5, 12 h or 1, 2, 3, 5, 7, 9 or 21 days after removal of vascular clips. Pancreatic blood flow (PBF), plasma lipase, interleukin-1beta (IL-1beta), interleukin-10, pancreatic cells proliferation and morphological signs of pancreatitis were determined. Pancreatic presence of fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta type II receptor (TGFbeta RII) was detected by immunohistochemisty. RESULTS: Exposure to I/R led to the development of acute necrotizing pancreatitis followed by regeneration. Morphological features showed maximal pancreatic damage between the 1(st) and 2(nd) day of reperfusion. It was correlated with a maximal increase in plasma lipase, and pro-inflammatory IL-1beta concentration, as well as, a reduction in PBF and pancreatic DNA synthesis. I/R increased FGF-2 content in pancreatic acinar cells between the 12(th) and 24(th) h, and between 5(th) and 9(th) day of reperfusion. At the 2(nd) day the presence of FGF-2 in pancreatic acinar cells was reduced. After I/R PDGF-A appeared in pancreatic vessels from the 12(th) h to 5 (th) day of reperfusion. PDGF-A was not observed in pancreatic acinar cells in the control or in I/R group. In pancreatic ducts, the presence of PDGF-A was reduced between the 1(st) and 3(rd), and between 7(th) and 9(th) day of reperfusion. In acinar cells, VEGF content was increased after I/R at the time between the 1(st) and 24(th) h, and between 3(rd) and 7(th) day of reperfusion. At the 2(nd) day of reperfusion, VEGF was not detected in the pancreatic acinar cells. Moreover, VEGF was found in the inflammatory infiltration, in the tubular complexes between the 2(nd) and 5(th) day, and in granulation tissue at the 9(th) day of reperfusion. In pancreatic acinar cells, I/R caused an increase in TGFbeta RII presence between the 5(th) and 24(th) h, and between 7(th) and 9(th) day of reperfusion. Between the 2(nd) and 5(th) day of reperfusion the acinar presence of TGFbeta RII was reduced. In the pancreatic ducts, the presence of TGFbeta RII was increased after I/R from the 1(st) h to 9(th) day of observation. Four weeks after induction of acute pancreatitis, the pancreatic regeneration was completed and the presence of growth factors tested returned to control value. CONCLUSIONS: The presence of FGF, VEGF, PDGF-A and TGFbeta RII is modified in the course of I/R-induced acute pancreatitis. Maximal content of FGF, VEGF and TGFbeta RII has been observed in early stage of pancreatic regeneration suggesting the involvement these factors in pancreatic recovery.     

Expression of angiotensin II receptors type 1 and type 2 in human preadipose cells during differentiation.

Human adipose tissue expresses all the components necessary for the production of angiotensin peptides. Although local effects of angiotensin II on cells from adipose tissue are beginning to be recognised, the expression of angiotensin receptors on human preadipocytes and adipocytes is still controversial. This study addresses the issue by monitoring the mRNA levels as well as the protein production of angiotensin II receptors of type 1 and 2 (AT 1 and AT 2 ) during differentiation of primary human preadipocytes in culture and in mature adipocytes. mRNA levels of the two receptor types are inversely correlated during adipose conversion. AT 1 receptor mRNA is greatly diminished within 12 days after induction of differentiation, while AT 2 receptor mRNA is elevated. mRNA levels of mature adipocytes confirm this trend. The regulation is not seen as strongly on the protein level. The amount of AT 2 receptor protein is increased, correlating well with the rise in specific glycerol-3-phosphate dehydrogenase activity of the cells, but the AT 1 receptor protein does not vary during the whole differentiation period. As the functional role of AT 2 receptors in adipose tissue is not known to date, further studies have to show if the AT 1 -mediated inhibitory actions on adipose conversion are downregulated in differentiating cells through decreased AT 1 /AT 2 receptor ratio.     

Retinoids potentiate transforming growth factor-β activity in bovine endothelial cells through up-regulating the expression of transforming growth factor-β receptors

Retinoic acid (RA) induces the activation of latent transforming growth factor-β (TGF-β) in bovine aortic endothelial cells (BAECs) via enhancement of cellular plasminogen activator (PA)/plasmin levels. The resultant TGF-β suppresses the excessive fibrinolytic activity by decreasing PA expression and stimulating expression of the PA inhibitor, PA inhibitor-1 (PAI-1), and inhibits cell proliferation. Here, we report that, in this regulatory system, RA simultaneously up-regulates the expression of TGF-β receptor types I and II, resulting in enhancement of TGF-β activity in the cells. RA increased the numbers of high- and low-affinity binding sites for ¹²⁵I-TGF-β1 2.1-fold and 1.5-fold, respectively, without alteration of their Kd values. Affinity labeling and Western and Northern blotting studies showed that, following RA treatment, surface levels of both type I and type II receptors increased due to augmentation in their mRNA levels. The effect was dose- and time-dependent. Treatment with 1 μM RA for 15 hr increased mRNA levels of type I and II receptor threefold and eightfold, respectively. Pretreatment of BAECs with either RA or retinol lowered the concentration of TGF-β1 required to suppress PA levels, to enhance PAI-1 levels, and to inhibit cell proliferation. Thus, retinoids may regulate cellular functions of BAECs not only by inducing the formation of active TGF-β but also by stimulating TGF-β receptor expression. This regulatory mechanism may sustain TGF-β-mediated regulation of EC function at a focal site where RA is acting. J. Cell. Physiol. 176:565–573, 1998. © 1998 Wiley-Liss, Inc.     

Type II epithelial cells are critical target for hyperoxia-mediated impairment of postnatal lung development

Type II epithelial cells are essential for lung development and remodeling, as they are precursors for type I cells and can produce vascular mitogens. Although type II cell proliferation takes place after hyperoxia, it is unclear why alveolar remodeling occurs normally in adults whereas it is permanently disrupted in newborns. Using a line of transgenic mice whose type II cells could be identified by their expression of enhanced green fluorescent protein and endogenous expression of surfactant proteins, we investigated the age-dependent effects of hyperoxia on type II cell proliferation and alveolar repair. In adult mice, type II cell proliferation was low during room air and hyperoxia exposure but increased during recovery in room air and then declined to control levels by day 7. Eight weeks later, type II cell number and alveolar compliance were indistinguishable from those in room air controls. In newborn mice, type II cell proliferation markedly increased between birth and postnatal day 7 before declining by postnatal day 14. Exposure to hyperoxia between postnatal days 1 and 4 inhibited type II cell proliferation, which resumed during recovery and was aberrantly elevated on postnatal day 14. Eight weeks later, recovered mice had 70% fewer type II cells and 30% increased lung compliance compared with control animals. Recovered mice also had higher levels of T1alpha, a protein expressed by type I cells, with minimal changes detected in genes expressed by vascular cells. These data suggest that perinatal hyperoxia adversely affects alveolar development by disrupting the proper timing of type II cell proliferation and differentiation into type I cells.     

Angiotensin AT1 receptor-associated protein Arap1 in the kidney vasculature is suppressed by angiotensin II

Arap1 is a protein that interacts with angiotensin II type 1 (AT(1)) receptors and facilitates increased AT(1) receptor surface expression in vitro. In the present study, we assessed the tissue localization and regulation of Arap1 in vivo. Arap1 was found in various mouse organs, with the highest expression in the heart, kidney, aorta, and adrenal gland. Renal Arap1 protein was restricted to the vasculature and to glomerular mesangial cells and was absent from tubular epithelia. A similar localization was found in human kidneys. To test the hypothesis that angiotensin II may control renal Arap1 expression, mice were subjected to various conditions to alter the activity of the renin-angiotensin system. A high-salt diet (4% NaCl, 7 days) upregulated Arap1 expression in mice by 47% compared with controls (0.6% NaCl, P = 0.03). Renal artery stenosis (7 days) or water restriction (48 h) suppressed Arap1 levels compared with controls (-64 and -62% in the clipped and contralateral kidney, respectively; and -50% after water restriction, P < 0.01). Angiotensin II infusion (2 μg·kg(-1)·min(-1), 7 days) reduced Arap1 mRNA levels compared with vehicle by 29% (P < 0.01), whereas AT(1) antagonism (losartan, 30 mg·kg(-1)·day(-1), 7 days) enhanced Arap1 mRNA expression by 52% (P < 0.01); changes in mRNA were paralleled by Arap1 protein abundance. Experiments with hydralazine and epithelial nitric oxide synthase-/- mice further suggested that Arap1 expression changed in parallel with angiotensin II, rather than with blood pressure per se. Similar to in vivo, Arap1 mRNA and protein were suppressed by angiotensin II in a time- and dose-dependent manner in cultured mesangial cells. In summary, Arap1 is highly expressed in the renal vasculature, and its expression is suppressed by angiotensin II. Thus Arap1 may serve as a local modulator of vascular AT(1) receptor function in vivo.     

Cell adhesion protein receptors as targets for transforming growth factor-beta action

Transforming growth factor-beta (TGF-beta) increases the incorporation of fibronectin and type I collagen into the extracellular matrix of fibroblasts and epithelial cells and enhances the attachment of thymocytes onto a fibronectin substratum. Investigation of the molecular basis for these effects showed that TGF-beta elevates specifically the expression of cell adhesion protein receptors. Treatment of cells with either form of TGF-beta, TGF-beta 1, or TGF-beta 2, increases the rate of receptor synthesis and the level of receptors on the cell surface. TGF-beta acts via two complementary mechanisms, elevation of receptor mRNA and faster kinetics of receptor beta subunit precursor to product conversion. The results show that the expression of cell adhesion receptors is susceptible to pretranslational and posttranslational regulation by factors that control cell morphology, proliferation, and differentiation such as TGF-beta.     

Regulation of human endometrial transforming growth factor beta1 and beta3 isoforms through menstrual cycle and medroxyprogesterone acetate treatment

The progesterone-induced differentiation of endometrial tissue from proliferative into secretory and decidua seems to be modulated by locally produced hormones and cytokines. Transforming growth factor beta (TGFbeta). a cytokine produced by endometrial cells, has been shown to modulate endometrial cell proliferation in vitro. Our aim was to evaluate the effects of medroxyprogesterone acetate (MPA) and the influence of menstrual cycle on the expression of TGFbeta1 and TGFbeta3 in human endometrium in vivo. In a double-blind, placebo-controlled trial, 46 healthy women with regular menstrual cycles received either MPA (10 mg/day) or placebo during 10 days. Endometrial and blood samples were collected 8-12 hours after the last MPA or placebo administration. Patients were classified into three groups according to biopsy dating and treatment: proliferative [tissue]/placebo, secretory [tissue]/placebo and secretory [tissue]/MPA. The immunohistochemical distribution of TGFbeta1 and TGFbeta1 mRNA was similar in all groups. Immunoreactive TGFbeta3 was present in the epithelium in 9.1% of proliferative samples, in 41.2% of secretory/placebo samples and in 87.5% of secretory/MPA samples (p=0.001). In the stroma, the frequency of TGFbeta3 staining was markedly increased after treatment with MPA (62.5%) compared to placebo (proliferative: 9.1%; secretory: 5.9%; p=0.005). The levels of TGFbeta3 mRNA increased during the secretory phase and were higher in the MPA-treated group, being directly correlated with morphological endometrial differentiation. It is concluded that MPA administration to healthy women increased TGFbeta3 but did not change TGFbeta1 gene and protein expression in the endometrium. This finding suggests that TGFbeta3 may be a local factor mediating progesterone- and progestogen-induced endometrial differentiation.     

T1alpha,a lung type I cell differentiation gene,is required for normal lung cell proliferation and alveolus formation at birth

T1alpha, a differentiation gene of lung alveolar epithelial type I cells, is developmentally regulated and encodes an apical membrane protein of unknown function. Morphological differentiation of type I cells to form the air-blood barrier starts in the last few days of gestation and continues postnatally. Although T1alpha is expressed in the foregut endoderm before the lung buds, T1alpha mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1alpha null mutant mice to study the role of T1alpha in lung development and differentiation and to gain insight into its potential function. Homozygous null mice die at birth of respiratory failure, and their lungs cannot be inflated to normal volumes. Distal lung morphology is altered. In the absence of T1alpha protein, type I cell differentiation is blocked, as indicated by smaller airspaces, many fewer attenuated type I cells, and reduced levels of aquaporin-5 mRNA and protein, a type I cell water channel. Abundant secreted surfactant in the narrowed airspaces, normal levels of surfactant protein mRNAs, and normal patterns and numbers of cells expressing surfactant protein-B suggest that differentiation of type II cells, also alveolar epithelial cells, is normal. Anomalous proliferation of the mesenchyme and epithelium at birth with unchanged numbers of apoptotic cells suggests that loss of T1alpha and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling.     

Nitric oxide induces TIMP-1 expression by activating the transforming growth factor beta-Smad signaling pathway

Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.     

Abrogation of TGFbeta signaling induces apoptosis through the modulation of MAP kinase pathways in breast cancer cells

Transforming growth factor beta (TGFbeta) can modulate the activity of various MAP kinases. However, how this pathway may mediate TGFbeta-induced malignant phenotypes remains elusive. We investigated the role of autocrine TGFbeta signaling through MAP kinases in the regulation of cell survival in breast carcinoma MCF-7 cells and untransformed human mammary epithelial cells (HMECs). Our results show that abrogation of autocrine TGFbeta signaling with the expression of a dominant negative type II TGFbeta receptor (DNRII) or the treatment with a TGFbeta type I receptor inhibitor significantly increased apoptosis in MCF-7 cell, but not in HMEC. The expression of DNRII markedly decreased activated/phosphorylated Erk, whereas increased activated/phosphorylated p38 in MCF-7 cells. In contrast, there was no or little change of phosphorylated Erk and p38 in HMECs after the expression of DNRII. Inhibition of Erk activity in MCF-7 control cell induced apoptosis whereas restoration of Erk activity in MCF-7 DNRII cell reduced apoptosis. Similarly, inhibition of p38 activity also inhibited apoptosis in MCF-7 DNRII cell. Thus, autocrine TGFbeta signaling can enhance the survival of MCF-7 cells by maintaining the level of active Erk high and the level of active p38 low. Furthermore, the survival properties of TGFbeta pathway appear related to transformation supporting the notion that it may be a potential target for cancer therapy.