Epithelial-mesenchymal Transdifferentiation Research Articles

Angiotensin II activates the Smad pathway during epithelial mesenchymal transdifferentiation

Epithelial to mesenchymal transdifferentiation is a novel mechanism that promotes renal fibrosis and here we investigated whether known causes of renal fibrosis (angiotensin II and transforming growth factor beta, TGFbeta) act through this pathway. We infused angiotensin II into rats for 1 day and found that it activated the Smad pathway which persisted for up to 2 weeks in chronically infused rats. Renal TGF-beta mRNA expression was increased at 3 days and its protein at 2 weeks suggesting Smad pathway activation occurred earlier than TGF-beta upregulation. In cultured human tubuloepithelial cells, angiotensin II caused a rapid activation of Smad signaling independent of TGF-beta however, Smad-dependent transcription after 1 day was TGF-beta mediated. Two weeks of angiotensin II infusion activated genes associated with epithelial mesenchymal transdifferentiation. Stimulation with angiotensin II for 3 days caused transdifferentiation of the cultured epithelial cells by TGF-beta-mediated processes; however, early changes were independent of endogenous TGF-beta. Smad7 overexpression, which blocks Smad2/3 activation, diminished angiotensin II-induced epithelial mesenchymal transdifferentiation. Our results show that angiotensin II activates the Smad signaling system by TGF-beta-independent processes, in vivo and in vitro, causing renal fibrosis.

Growth factors in benign prostatic hyperplasia: Basic science implications

Benign prostatic hyperplasia (BPH) is the most common proliferative disease of the prostate of men in the United States. The histopathology of BPH strongly implicates local paracrine and autocrine growth factors and inflammatory cytokines in its pathogenesis. A complex milieu of growth-regulatory proteins includes members of the fibroblast, insulin-like, and transforming growth factor families. It appears that these proteins and downstream effector molecules, in addition to a variety of interleukins, are overexpressed in BPH and, working together, create a landscape of increased stromal and epithelial growth and mesenchymal transdifferentiation that leads to disease progression. Inflammation, commonly present in BPH, may contribute to tissue injury, and cytokines produced by inflammatory cells may serve to drive local growth factor production and angiogenesis in the tissues as a "wound healing" response. As we begin to unravel the precise mechanisms involved, new treatments for BPH aimed at these interacting pathways may emerge.

Demonstration of Epithelial-Mesenchymal Transition in Kidney

Traditional Periodic Acid Schiff has been extensively used, coupled with immunohistochemistry for epithelia or mesenchymal cells, to highlight renal tubular basement membrane (TBM). We recently tried to perform such technique in a 5/6 nephrectomy model of progressive renal fibrosis to demonstrate TBM disruption as an evidence for epithelial-mesenchymal transdifferentiation. Despite excellent basement membrane staining with traditional fuchsin-Periodic Acid Schiff, the interface between epithelial and mesenchymal cells was frequently blurred when revealed with 3'3 diaminobenzidine tetrachloride-peroxidase. Also, it was inadequate when revealed with alkaline phosphatase-fast red. We devised a triple staining method with Periodic Acid-Thionin Schiff to highlight basement membrane in blue, after double immunostaining for epithelium and mesenchymal cells. Blue basement membrane rendered a brisk contrast and highlighted boundaries between epithelial-mesenchymal interfaces. This method was easy to perform and useful to demonstrate the TBM, yield a clear demonstration of the very focal TBM disruption found in this model of progressive renal fibrosis.

Rho Kinase Inhibition: A New Approach for Treating Diabetic Nephropathy?

Rho family GTPases have received increasing attention as critical regulators of cell function (1). Initially, they were shown to regulate actin dynamics, thereby modulating development, cell migration, immune responses, and cancer cell invasion and metastasis. More recent studies have shown that they are also involved in cell-cell adhesion and cell cycle progression. RhoA is one of the most widely studied of the 22 mammalian members of the family, and the serine-threonine kinase Rho kinase (ROCK) is a major RhoA effector. RhoA/ROCK has a number of functions in the kidney. RhoA/ROCK enhances Ca2+-dependent vascular smooth muscle contraction, thereby modulating tone (2–4). The potent vasoconstrictor angiotensin II (AngII) activates ROCK in smooth muscle cells. The ROCK inhibitors fasudil and Y-27632 dilate afferent and efferent arterioles and reverse AngII-dependent arteriolar vasoconstriction. At the cellular level, RhoA/ROCK mediates cytoskeletal rearrangement in renal tubule cells, mesangial cells and podocytes and may contribute to epithelial-mesenchymal transdifferentiation, which may be important in the development of renal fibrosis. ROCK inhibitors have renoprotective effects in a number of models of kidney damage (2). They prevent tubulointerstitial fibrosis following unilateral ureteral obstruction, and decrease structural and functional damage in hypertensive models without affecting blood pressure. When activated, RhoA translocates from the cytoplasm to the cell membrane. RhoA translocation to …

Transforming growth factor- -induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38 mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial-myofibroblast transdifferentiation

Transforming growth factor-beta (TGFbeta)-induced epithelial-myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of alpha-smooth muscle cell actin (alphaSMA) expression, a hallmark of myofibroblast formation, induced by TGFbeta in renal proximal tubular cells. Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The alphaSMA synthesis was assessed by western blotting or immunofluorescent staining of cellular alphaSMA. To assess the regulation of the alphaSMA promoter, tubular cells were transiently transfected with a 785 bp alphaSMA promoter-luciferase reporter construct and vectors interfering with the Smad pathway. Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFbeta-induced alphaSMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38beta but not of p38alpha potently inhibited alphaSMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFbeta effect, confirming the role of p38beta in the regulation of TGFbeta-induced alphaSMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFbeta-induced alphaSMA synthesis. TGFbeta-induced alphaSMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial-mesenchymal transdifferentiation.

Effect of Genistein on epithelial-mesenchymal transdifferentiation induced by transforming growth factor-β1 in human pancreatic cancer cell line Panc-1

AIM: To investigate the effect of Genistein on epithelial-mesenchymal transdifferentiation (EMT) induced by transforming growth factor-β1 (TGF-βl) in human pancreatic cancer cell line Panc-1, and to explore the mechanism of Genistein inhibiting invasion of Panc-1 cells. METHODS: Panc-1 cells were treated with TGF-β1 and Genistein (0, 1, 25, 50 μmol/L), and those treated with PSB served as controls. Tran-swell chamber assay was performed to determine the invasion ability change of Panc-1 cells. Reverse transcription-polymerase chain reaction (RT-PCR) was used to estimate the mRNA expression of vimentin and E-cadherin. Western bloting assay was used to measure the protein expression of E-cadherin. Cell structure was observed by microscopy. RESULTS: TGF-β1obviously promoted EMT and invasion abilibty of Pane-l cells. Not only the invasion ability but also EMT induced by TGF-β1 were significantly inhibited by Genistein in a dose-dependent manner. The number of Pane-l cells was larger in 0 μmol/L Genistein group than that in the control group (99.16±11.30 vs 65.46±8.99, P＜0.05). Genistein at concentraion of 50 μmol/L down-regulated the mRNA expression of vimentin and up-regulated the mRNA and protein expression of E-cadherin. The characteristic morphology of EMT was reversed. CONCLUSION: Genistein can inhibit TGF-β1-induced invasion of Pane-1 cells remarkably, which may be one of its anti-invasion mechanisms.

Activation of the Renin-Angiotensin System and Chronic Hypoxia of the Kidney

Recent studies emphasize the role of chronic hypoxia in the kidney as a final common pathway to end-stage renal failure (ESRD). Hypoxia of tubular cells leads to apoptosis or epithelial-mesenchymal transdifferentiation, which in turn exacerbates the fibrosis of the kidney with the loss of peritubular capillaries and subsequent chronic hypoxia, setting in train a vicious cycle whose end-point is ESRD. While fibrotic kidneys in an advanced stage of renal disease are devoid of peritubular capillary blood supply and oxygenation to the corresponding region, imbalances in vasoactive substances can cause chronic hypoxia even in the early phase of kidney disease. Among various vasoactive substances, local activation of the renin-angiotensin system (RAS) is particularly important because it can lead to the constriction of efferent arterioles, hypoperfusion of postglomerular peritubular capillaries, and subsequent hypoxia of the tubulointerstitium in the downstream compartment. In addition, angiotensin II induces oxidative stress via the activation of NADPH oxidase. Oxidative stress damages endothelial cells directly, causing the loss of peritubular capillaries, and also results in relative hypoxia due to inefficient cellular respiration. Thus, angiotensin II induces renal hypoxia via both hemodynamic and nonhemodynamic mechanisms. In the past two decades, considerable gains have been realized in retarding the progression of chronic kidney disease by emphasizing blood pressure control and blockade of the RAS. Chronic hypoxia in the kidney is an ideal therapeutic target, and the beneficial effects of blockade of RAS in kidney disease are, at least in part, mediated by the amelioration of local hypoxia. (Hypertens Res 2008; 31: 175-184).

The inhibitory effects of Disabled-2 (Dab2) on Wnt signaling are mediated through Axin

beta-Catenin-mediated Wnt signaling is essential in embryonic development and in adult tissues. Recent studies have demonstrated that Axin not only plays an important inhibitory role in coordinating beta-catenin degradation, but is itself degraded by the low-density-lipoprotein receptor-related protein (LRP)5/6 Wnt co-receptor. Here, we demonstrate that the endocytic adaptor molecule Disabled-2 (Dab2), which we have previously demonstrated to act as an inhibitor of beta-catenin signaling, interacts with Axin and prevents its interaction with and degradation by the LRP5 co-receptor, thereby increasing its half-life and stabilization. Dab2 levels induced during retinoic acid-induced differentiation of F9, or during transforming growth factor-beta-induced epithelial-mesenchymal transdifferentiation of mouse mammary epithelial cells result in the stabilization of Axin and concomitant inhibition of beta-catenin signaling. Ectopic expression of Dab2 in F9 cells as well as in transformed cell lines results in increased Axin expression and attenuation of Wnt-mediated signaling. We conclude that Dab2 may play an important role in the maintenance of the differentiated state and restrain Wnt-mediated proliferation through its association with and modulation of Axin.

Ionising radiation induces changes associated with epithelial-mesenchymal transdifferentiation and increased cell motility of A549 lung epithelial cells

Radiotherapy remains a major therapeutic option for patients with advanced lung cancer. Nevertheless, the effects of irradiation on malignant biological behaviours (e.g. migration and transformation of cancer cells) have yet to be clarified. We conducted an in vitro study to investigate the radiation-induced alterations including morphology, adhesion, and cell motility of A549 human lung cancer cells. These changes, which are associated with epithelial-mesenchymal transdifferentiation (EMT), seem to be linked to radiation-induced fibrosis, which represents one of the most common long-term adverse effects of curative radiotherapy. In addition, loss of intercellular adhesion and increased cell motility may be involved in post-radiotherapy-associated metastasis. We showed that stress fibres and focal adhesions are increased and that cell–cell junctions are decreased in response to ionising radiation. Radiation also significantly increased cell motility. The p38-specific inhibitor, SB203580, reduced the radiation-promoted migration of A549 cells, whereas SP600125, a JNK MAPK-specific inhibitor, inhibited both inherent and radiation-mediated cell motility. Consistent with this observation, radiation up-regulated the phosphorylation of p38 MAPK. Current approaches to cancer treatment involving more intensive radiotherapy regimens have been suggested to be associated with a higher incidence of local or distant metastasis. Therefore, a subset of patients may benefit from a combination of radiotherapy with inhibitors of EMT or cell migration.

Wen‐pi‐tang‐Hab‐Wu‐ling‐san, an oriental herbal prescription, attenuates epithelial‐mesenchymal transdifferentiation stimulated by TGF‐β1 in kidney cells

Wen-pi-tang-Hab-Wu-ling-san (WHW), an oriental herbal prescription, is currently used in oriental clinics for the treatment of chronic renal failure (CRF). While its effectiveness has been supported by a series of modern studies, the underlying mechanism remains poorly understood. CRF progression involves tubulointerstitial fibrosis where transforming growth factor-beta1 (TGF-beta1) plays a critical role by inducing epithelial-mesenchymal transdifferentiation (EMT). This study examined whether WHW extract attenuated the TGF-beta1-induced EMT in Madin-Darby canine kidney cells. When the cells were stimulated by TGF-beta1 (2.5 ng/mL), they exhibited an elongated, spindle-shaped appearance but this morphological change was significantly suppressed by WHW extract (1 mg/mL). The WHW extract did not show notable cytotoxicity and even mitigated the cytotoxic effects of TGF-beta1. It inhibited the expression of alpha-smooth muscle actin (alpha-SMA), a marker of EMT, but not the secretion of matrix metalloproteinases stimulated by TGF-beta1. The WHW extract also inhibited the phosphorylation of Smad2 that mediates TGF-beta1 signaling leading to alpha-SMA expression. The present study suggests that WHW extract may provide renal protective effects through modulation of the TGF-beta1/Smad2/alpha-SMA pathway involved in fibrosis.

Epithelial-mesenchymal transdifferentiation of bile duct cells during fibrogenesis due to cholestasis

Epithelial-mesenchymal transdifferentiation of bile duct cells during fibrogenesis due to cholestasis

JNK mediates TGF-β1-induced epithelial mesenchymal transdifferentiation of mouse transformed keratinocytes

In this study we analyzed the role of the c-Jun N-terminal kinases (JNK) pathway in the TGF-β1 stimulation of urokinase-type plasminogen activator (uPA), initial stages of epithelial-mesenchymal transdifferentiation (EMT) and cell migration. TGF-β1 induces JNK phosphorylation, c-Jun transactivation and AP1 activation. The involvement of JNK was evaluated using dominant negative mutants SEK-1 AL, JNK and cJun, depletion of JNK1,2 proteins by treatment of cells with antisense oligonucleotides, as well as the chemical inhibitor SP600125. Our results demonstrated that the JNK pathway is required in the TGF-β1 enhancement of uPA, fibronectin, E-cadherin delocalization, actin re-organization and vimentin expression, concomitant with the induction of cell migration. These results allow us to suggest a role of JNK in the TGF-β1 induction of EMT in relation with the stimulation of malignant properties of mouse transformed keratinocytes.

Adenovirus-Based Transient Expression Systems for Peritoneal Membrane Research

Peritoneal membrane research has provided important insights into the physiology and pathophysiology of this tissue that is of vital importance for peritoneal dialysis patients. Among the various tools and methodologies used to study the peritoneum, we have extensively used adenovirus-mediated gene transfer. A literature review was carried out. Information from reviewed papers was combined with the authors' experience and results. We have used first-generation adenoviruses that are simple to construct and can infect a wide range of dividing and nondividing cell types. These vectors are restricted, however, in that they provide only a short duration of transgene expression and may elicit an inflammatory response. Modifications to this technology with helper-dependent adenovirus may circumvent these problems but with increased complexity of construction. Adenovirus-mediated gene transfer has been used to evaluate the effect of several cytokines and growth factors on peritoneal membrane physiology. We have used intraperitoneal delivery of transforming growth factor-beta to generate an experimental model system of resolving peritoneal fibrosis and epithelial mesenchymal transdifferentiation. We have studied the effects of the inflammatory cytokines interleukin-1beta and tumor necrosis factor alpha on the peritoneum, and have shown that antiangiogenic factors such as sFLT-1 and angiostatin can reduce the damaging effects of exposure to peritoneal dialysis solutions in an animal model. The use of recombinant adenoviruses to genetically modify cells and tissues is now a common laboratory research tool. This technique has provided important advances in our understanding of the peritoneal membrane.

Caveolin-1 is up-regulated in transdifferentiated lens epithelial cells but minimal in normal human and murine lenses

Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types. Caveolae play a role in lipid transport, endocytosis, signal transduction, and cell transformation. Expression of caveolin-1, the principal component of caveolae and a regulator of caveolae-dependent signaling and endocytosis, was investigated in lens epithelial cells and lens fiber cells in wild-type (wt) and SPARC-null mice and normal human donors in vivo and in vitro. RT-PCR, immunofluorescence and immunoblot analyses of human and murine ocular tissues revealed that caveolin-1 was expressed in the corneal epithelium, corneal endothelial cells, and blood vessels of iris, ciliary body and retina, but minimal in the normal lens epithelia or fiber cells of murine and human lens. In contrast, caveolin-1 was significantly up-regulated in mesenchymal-transdifferentiated lens epithelia in SPARC-null cataract lenses. In addition, lens epithelial cells from primary culture or from cultures of immortalized lens epithelial cell lines expressed significant amounts of caveolin-1. The lens epithelial cells expressed epidermal growth factor (EGF) receptor and were responsive to EGF-mediated cell proliferation, but they did not show EGF-dependent caveolin-1 tyrosine phosphorylation. Caveolin-1 might have a role in the process of epithelial-mesenchymal transdifferentiation (EMT) in the lens, the most common cause of vision loss in human secondary cataracts.

Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion

Malformations in secondary palate fusion will lead to cleft palate, a common human birth defect. Palate fusion involves the formation and subsequent degeneration of the medial edge epithelial seam. The cellular mechanisms underlying seam degeneration have been a major focus in the study of palatogenesis. Three mechanisms have been proposed for seam degeneration: lateral migration of medial edge epithelial cells; epithelial-mesenchymal trans-differentiation; and apoptosis of medial edge epithelial cells. However, there is still a great deal of controversy over these proposed mechanisms. In this study, we established a [Rosa26<-->C57BL/6] chimeric culture system, in which a Rosa26-originated ;blue' palatal shelf was paired with a C57BL/6-derived ;white' palatal shelf. Using this organ culture system, we observed the migration of medial edge epithelial cells to the nasal side, but not to the oral side. We also observed an anteroposterior migration of medial edge epithelial cells, which may play an important role in posterior palate fusion. To examine epithelial-mesenchymal transdifferentiation during palate fusion, we bred a cytokeratin 14-Cre transgenic line into the R26R background. In situ hybridization showed that the Cre transgene is expressed exclusively in the epithelium. However, beta-galactosidase staining gave extensive signals in the palatal mesenchymal region during and after palate fusion, demonstrating the occurrence of an epithelial-mesenchymal transdifferentiation mechanism during palate fusion. Finally, we showed that Apaf1 mutant mouse embryos are able to complete palate fusion without DNA fragmentation-mediated programmed cell death, indicating that this is not essential for palate fusion in vivo.

The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFbeta1-Smad (transforming growth factor-beta1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFbeta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFbeta1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, alpha-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFbeta1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGFbeta1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFbeta1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

TGF-β1-induced EMT can occur independently of its proapoptotic effects and is aided by EGF receptor activation

Apoptosis and epithelial-mesenchymal transdifferentiation (EMT) occur in stressed tubular epithelial cells and contribute to renal fibrosis. Transforming growth factor (TGF)-beta(1) promotes these responses and we examined whether the processes were interdependent in vitro. Direct (caspase inhibition) and indirect [epidermal growth factor (EGF) receptor stimulation] strategies were used to block apoptosis during TGF-beta(1) stimulation, and the subsequent effect on EMT was assessed. HK-2 cells were exposed to TGF-beta(1) with or without preincubation with ZVAD-FMK (pan-caspase inhibitor) or concomitant treatment with EGF plus or minus preincubation with LY-294002 (PI3-kinase inhibitor). Cells were then assessed for apoptosis and proliferation by flow cytometry, crystal violet assay, and Western blotting. Markers of EMT were assessed by microscopy, immunofluorescence, real-time RT-PCR, Western blotting, PAI-1 reporter assay, and collagen gel contraction assay. TGF-beta(1) caused apoptosis and priming for staurosporine-induced apoptosis. This was blocked by ZVAD-FMK. However, ZVAD-FMK did not prevent EMT following TGF-beta(1) treatment. EGF inhibited apoptosis and facilitated TGF-beta(1) induction of EMT by increasing proliferation and accentuating E-cadherin loss. Additionally, EGF significantly enhanced TGF-beta(1)-induced collagen I gel contraction. EGF increased Akt phosphorylation during EMT, and the prosurvival effect of this was confirmed using LY-294002, which reduced EGF-induced Akt phosphorylation and reversed its antiapoptotic and proproliferatory effects. TGF-beta(1) induces EMT independently of its proapoptotic effects. TGF-beta(1) and EGF together lead to EMT. EGF increases proliferation and resistance to apoptosis during EMT in a PI3-K Akt-dependent manner. In vivo, EGF receptor activation may assist in the selective survival of a transdifferentiated, profibrotic cell type.

Chronic Hypoxia and Tubulointerstitial Injury

Recent studies emphasize the role of chronic hypoxia in the tubulointerstitium as a final common pathway to end-stage renal failure. When advanced, tubulointerstitial damage is associated with the loss of peritubular capillaries. Associated interstitial fibrosis impairs oxygen diffusion and supply to tubular and interstitial cells. Hypoxia of tubular cells leads to apoptosis or epithelial-mesenchymal transdifferentiation. This in turn exacerbates fibrosis of the kidney and subsequent chronic hypoxia, setting in train a vicious cycle whose end point is ESRD. A number of mechanisms that induce tubulointerstitial hypoxia at an early stage have been identified. Glomerular injury and vasoconstriction of efferent arterioles as a result of imbalances in vasoactive substances decrease postglomerular peritubular capillary blood flow. Angiotensin II not only constricts efferent arterioles but, via its induction of oxidative stress, also hampers the efficient utilization of oxygen in tubular cells. Relative hypoxia in the kidney also results from increased metabolic demand in tubular cells. Furthermore, renal anemia hinders oxygen delivery. These factors can affect the kidney before the appearance of significant pathologic changes in the vasculature and predispose the kidney to tubulointerstitial injury. Therapeutic approaches that target the chronic hypoxia should prove effective against a broad range of renal diseases. Current modalities include the improvement of anemia with erythropoietin, the preservation of peritubular capillary blood flow by blockade of the renin-angiotensin system, and the use of antioxidants. Recent studies have elucidated the mechanism of hypoxia-induced transcription, namely that prolyl hydroxylase regulates hypoxia-inducible factor. This has given hope for the development of novel therapeutic approaches against this final common pathway.

TGF‐β control of cell proliferation

This article focuses on recent findings that the type V TGF-beta receptor (TbetaR-V), which co-expresses with other TGF-beta receptors (TbetaR-I, TbetaR-II, and TbetaR-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-beta and that TGF-beta activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-beta is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-beta controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent TbetaR-I/TbetaR-II/Smad2/3/4 signaling cascade, are required for mediating TGF-beta-induced growth inhibition. Recent studies revealed that TbetaR-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-beta-induced growth inhibition in concert with TbetaR-I and TbetaR-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the TbetaR-V-mediated growth inhibitory signaling cascade. The TbetaR-V signaling cascade appears to cross-talk with the TbetaR-I/TbetaR-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the TbetaR-V signaling cascade may enable carcinoma cells to escape from TGF-beta growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-beta binding to TbetaR-II and TbetaR-I is a signal controlling TGF-beta partitioning between two distinct endocytosis pathways and resultant TGF-beta responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-beta-induced cellular growth inhibition, cross-talk between the TbetaR-V and other signaling cascades, the signal that controls TGF-beta responsiveness and the role of TbetaR-V in tumorigenesis.

Diverse Biological Effect and Smad Signaling of Bone Morphogenetic Protein 7 in Prostate Tumor Cells

We found that bone morphogenetic protein (BMP) 7, a member of the BMP family, was strikingly up-regulated during the development of primary prostatic adenocarcinoma in the conditional Pten deletion mouse model. To determine the relevance of this finding to human prostate cancer, we examined the expression of BMPs and BMP receptors (BMPR) as well as the responsiveness to recombinant human BMP7 in a series of human prostate tumor cell lines. All prostatic cell lines tested expressed variable levels of BMP2, BMP4, and BMP7 and at least two of each type I and II BMPRs. In all cases, BMP7 induced Smad phosphorylation in a dose-dependent manner, with Smad5 activation clearly demonstrable. However, the biological responses to BMP7 were cell type specific. BPH-1, a cell line representing benign prostatic epithelial hyperplasia, was growth arrested at G1. In the bone metastasis-derived PC-3 prostate cancer cells, BMP7 induced epithelial-mesenchymal transdifferentiation with classic changes in morphology, motility, invasiveness, and molecular markers. Finally, BMP7 inhibited serum starvation-induced apoptosis in the LNCaP prostate cancer cell line and more remarkably in its bone metastatic variant C4-2B line. Each of the cell lines influenced by BMP7 was also responsive to BMP2 in a corresponding manner. The antiapoptotic activity of BMP7 in the LNCaP and C4-2B cell lines was not associated with a significant alteration in the levels of the proapoptotic protein Bax or the antiapoptotic proteins Bcl-2, Bcl-xl, and X-linked inhibitor of apoptosis. However, in C4-2B cells but not in LNCaP cells, a starvation-induced decrease in the level of survivin was counteracted by BMP7. Taken together, these findings suggest that BMPs are able to modulate the biological behavior of prostate tumor cells in diverse and cell type-specific manner and point to certain mechanisms by which these secreted signaling molecules may contribute to prostate cancer growth and metastasis.