Primary cilia control the maturation of tubular lumen in renal collecting duct epithelium.

The key role of the primary cilium in developmental processes is illustrated by ciliopathies resulting from genetic defects of its components. Ciliopathies include a large variety of dysmorphic syndromes that share in common the presence of multiple kidney cysts. These observations suggest that primary cilia may control morphogenetic processes in the developing kidney. In this study, we assessed the role of primary cilium in branching tubulogenesis and/or lumen development using kidney collecting duct-derived mCCDN21 cells that display spontaneous tubulogenic properties when grown in collagen-Matrigel matrix. Tubulogenesis and branching were not altered when cilium body growth was inhibited by Kif3A or Ift88 silencing. In agreement with the absence of a morphogenetic effect, proliferation and wound-healing assay revealed that neither cell proliferation nor migration were altered by cilium body disruption. The absence of cilium following Kif3A or Ift88 silencing in mCCDN21 cells did not alter the initial stages of tubular lumen generation while lumen maturation and enlargement were delayed. This delay in tubular lumen maturation was not observed after Pkd1 knockdown in mCCDN21 cells. The delayed lumen maturation was explained by neither defective secretion or increased reabsorption of luminal fluid. Our results indicate that primary cilia do not control early morphogenetic processes in renal epithelium. Rather, primary cilia modulate tubular lumen maturation and enlargement resulting from luminal fluid accumulation in tubular structures derived from collecting duct cells.

Spatially restricted hyaluronan production by Has2 drives epithelial tubulogenesis in vitro.

Generation of branched tubes from an epithelial bud is a fundamental process in development. We hypothesized that induction of hyaluronan synthase (Has) and production of hyaluronan (HA) drives tubulogenesis in response to morphogenetic cytokines. Treatment of J3B1A mammary cells with transforming growth factor-ß1 or renal MDCK and mCCD-N21 cells with hepatocyte growth factor induced strong and specific expression of Has2. Immunostaining revealed that HA was preferentially produced at the tips of growing tubules. Inhibition of HA production, either by 4-methylumbelliferone (4-MU) or by Has2 mRNA silencing, abrogated tubule formation. HA production by J3B1A and mCCD-N21 cells was associated with sustained activation of ERK and S6 phosphorylation. However, silencing of either CD44 or RHAMM (receptor for HA-mediated motility), the major HA receptors, by RNA interference, did not alter tubulogenesis, suggesting that this process is not receptor-mediated.

Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Inhibition of basal p38 or JNK activity enhances epithelial barrier function through differential modulation of claudin expression.

Tight junctions (TJs) form a barrier to the paracellular diffusion of ions and solutes across epithelia. Although transmembrane proteins of the claudin family have emerged as critical determinants of TJ permeability, little is known about the signaling pathways that control their expression. The aim of this study was to assess the role of three mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH(2)-terminal kinases (JNKs), and p38 kinases, in the regulation of epithelial barrier function and claudin expression in mammary epithelial cells. Addition of either PD169316 (a p38 inhibitor) or SP600125 (a JNK inhibitor) induced formation of domes (a phenomenon dependent on TJ barrier function) and enhanced transepithelial electrical resistance, whereas U0126 (an inhibitor of the ERK1/2 activators MEK1/MEK2) had no significant effect. Similar results were obtained using mechanistically unrelated p38 or JNK inhibitors. PD169316 increased the expression of claudin-4 and -8, whereas SP600125 increased claudin-4 and -9 and downregulated claudin-8. Silencing of p38alpha by isoform-specific small interfering RNAs increased claudin-4 and -8 mRNAs, whereas silencing of p38beta only increased claudin-4 mRNA. Silencing of either JNK1 or JNK2 increased claudin-9 mRNA expression while decreasing claudin-8 mRNA. Moreover, selective silencing of JNK2 increased claudin-4 and -7 mRNAs. Finally, both PD169316 and SP600125 inhibited the paracellular diffusion of Na(+) and Cl(-) across epithelial monolayers. Collectively, these results provide evidence that inhibition of either p38 or JNK enhances epithelial barrier function by selectively modulating claudin expression, implying that the basal activity of these MAPKs exerts a tonic effect on TJ ionic permeability.

Unsaturated fatty acids promote hepatoma proliferation and progression through downregulation of the tumor suppressor PTEN.

BACKGROUND/AIMS: The impact of dietary fatty acids on the development of cancers is highly controversial. We recently demonstrated that unsaturated fatty acids trigger the downregulation of the tumor suppressor PTEN through an mTOR/NF-kappaB-dependent mechanism in hepatocytes. In this study, we investigated whether unsaturated fatty acids promote hepatoma progression by downregulating PTEN expression. METHODS: The effects of fatty acids and PTEN-specific siRNAs on proliferation, invasiveness and gene expression were assessed using HepG2 hepatoma cells. The tumor promoting activity of unsaturated fatty acids was evaluated in vivo using HepG2 xenografts in nude mice. RESULTS: Incubation of HepG2 cells with unsaturated fatty acids, or PTEN-specific siRNAs, increased cell proliferation, cell migration and invasiveness, and altered the expression of genes involved in inflammation, epithelial-to-mesenchymal transition and carcinogenesis. These effects were dependent on PTEN expression levels and were prevented by mTOR and NF-kappaB inhibitors. Consistent with these data, the development and size of subcutaneous HepG2-derived tumors in nude mice xenografts were dramatically increased when mice were fed with an oleic acid-enriched diet, even in the absence of weight gain. CONCLUSIONS: These data demonstrate that dietary unsaturated fatty acids promote hepatoma progression by reducing the expression of the tumor suppressor PTEN.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

Raf kinase inhibitor protein positively regulates cell-substratum adhesion while negatively regulating cell-cell adhesion.

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.

Oncostatin M-induced effects on EMT in human proximal tubular cells: differential role of ERK signaling.

Growing evidence suggests that a proportion of interstitial myofibroblasts detected during renal tubulointerstitial fibrosis originates from tubular epithelial cells by a process called epithelial-mesenchymal transition (EMT). The IL-6-type cytokine oncostatin M (OSM) has been recently implicated in the induction of EMT. We investigated OSM effects on the expression of both cell-cell contact proteins and mesenchymal markers and studied OSM-induced intracellular signaling mechanisms associated with these events in human proximal tubular cells. Human recombinant OSM attenuated the expression of N-cadherin, E-cadherin, and claudin-2 in human kidney-2 (HK-2) cells associated with the induction of HK-2 cell scattering in 3D collagen matrices. Conversely, expression of collagen type I, vimentin, and S100A4 was induced by OSM. OSM-stimulated cell scattering was inhibited by antibodies against gp130. Besides inducing phosphorylation of Stat1 and Stat3, OSM led to a strong concentration- and time-dependent phosphorylation of the mitogen-activated protein kinases ERK1, ERK2, and ERK5. MEK1/2 inhibitor U0126 (10 muM) blocked basal and OSM-induced ERK1/2 phosphorylation but not phosphorylation of either ERK5 or Stat1/3. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at concentrations which inhibit ERK1/2 phosphorylation but not ERK5 phosphorylation, restored N-cadherin expression in the presence of OSM, inhibited basal claudin-2 expression, but did not affect either basal or OSM-inhibited E-cadherin expression or OSM-induced expression of collagen type I and vimentin. These results suggest that in human proximal tubular cells ERK1/2 signaling represents an important component of OSM's inhibitory effect on N-cadherin expression. Furthermore, functional ERK1/2 signaling is necessary for basal claudin-2 expression.

Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells.

BACKGROUND: Formation of branching tubes is a fundamental step in the development of glandular organs. To identify extracellular cues that orchestrate epithelial tubulogenesis, we employed an in vitro assay in which EpH4-J3B1A mammary epithelial cells form spheroidal cysts when grown in collagen gels under serum-free conditions, but form branching tubules in the presence of fetal calf serum (FCS). RESULTS: Initial experiments showed that the tubulogenesis-inducing activity of FCS was markedly increased by heating (70 degrees C) or transient acidification to pH3. We therefore hypothesized that the tubulogenic agent was transforming growth factor-beta (TGF-beta), a cytokine that is present in serum in latent form and can be activated by heat or acid treatment. We found indeed that the tubulogenic activity of acidified FCS is abrogated by addition of either SB-431542, a selective inhibitor of the TGF-beta type I receptor, or a neutralizing antibody to TGF-beta-1. On the other hand, addition of low concentrations (20-100 pg/ml) of exogenous TGF-beta-1 recapitulated the effect of acidified FCS in inducing morphogenesis of hollow tubes. In contrast, higher concentrations of TGF-beta-1 induced the formation of thin cellular cords devoid of a detectable lumen. To gain insight into the mechanisms underlying TGF-beta-1-induced tube formation, we assessed the potential role of matrix metalloproteinases (MMPs). By western blot and gelatin zymography, we observed a dose-dependent increase in MMP-9 upon TGF-beta-1 treatment. Tube formation was suppressed by a synthetic broad-spectrum metalloproteinase inhibitor, by recombinant tissue inhibitor of metalloproteinases-2 (TIMP-2) and by a selective inhibitor of MMP-9, indicating that this morphogenetic process requires the activity of MMP-9. CONCLUSION: Altogether, our results provide evidence that, at low concentrations, TGF-beta-1 promotes MMP-dependent branching tubulogenesis by mammary epithelial cells in vitro, and suggest that it plays a similar role during mammary gland development in vivo.

Bone morphogenetic protein-4 abrogates lumen formation by mammary epithelial cells and promotes invasive growth.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that regulate key developmental processes, but are also overexpressed in many carcinomas. To assess whether BMPs would influence the three-dimensional architecture of epithelial structures, we took advantage of an in vitro model in which mammary epithelial cells form alveolar-like spherical cysts in collagen gels. We found that BMP-4 has a dramatic, biphasic effect on the organization of epithelial cysts. When added in the concentration range of 1-10 ng/ml, the cytokine abrogates lumen formation and induces the outgrowth of multiple invasive cord-like structures. At higher concentrations (20-100 ng/ml), BMP-4 additionally disrupts cell-cell adhesion, resulting in cyst disintegration and scattering of individual cells into the surrounding collagen matrix. The finding that BMP-4 subverts the ability of mammary epithelial cells to form polarized lumen-containing structures and endows them with invasive properties supports the involvement of this cytokine in the progression of breast cancer.

A role for atm in E-cadherin-mediated contact inhibition in epithelial cells.

Ataxia telangiectasia is a hereditary pleiomorphic syndrome caused by loss of Atm, a phosphoprotein involved in multiple signaling pathways. Here, we propose a novel role for atm in cultured epithelial cells, namely the regulation of cell growth by contact inhibition. We show that atm is upregulated in epithelial cells reaching confluence. Conditional expression of the PI 3-Kinase domain of atm in non-confluent Tac-2 epithelial cells increases the expression of the anti-proliferative gene Tis-21 and downregulates key cell cycle regulator genes, such as cyclins A, B1, B2, E and E2. Finally, we demonstrate that upregulation of atm, and thus Tis-21, in confluent Tac-2 cells can be inhibited by an E-cadherin antibody blocking specifically homophilic E-cadherin interactions between adjacent cell surfaces. Altogether, these results suggest that atm could participate in a molecular pathway linking extracellular signalling to cell cycle control and may help further clarify the role of Atm in epithelial cell biology and carcinogenesis.

Bovine microvascular endothelial cells immortalized with human telomerase.

Primary cultures of bovine microvascular endothelial cells (BME) isolated from the adrenal cortex, are commonly used to study vascular endothelium, but have a limited life span. To circumvent these limitations, we have immortalized BME cells with either simian virus 40 (SV40) or with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have investigated whether the clonal populations obtained, maintain differentiated properties characteristic of microvascular endothelium. Immortalized cells were characterized for maintenance of typical endothelial morphology, marker expression, and functional characteristics including uptake of Acetylated low-density lipoprotein (Ac-LDL), capillary-like tube formation in three-dimensional collagen gels, as well as metalloproteinase (MMP) and plasminogen activator (PA)-mediated extracellular proteolysis. Whilst immortalization of BME cells with SV40 was associated with loss of endothelial-specific properties, hTERT-BME exhibited an endothelial phenotype similar to that of wild-type endothelial cells. Specifically, they showed a typical cobblestone morphology, were contact-inhibited, expressed endothelial cell-specific markers (e.g., CD31, vWF) and both fibroblast growth factor receptor 1 (FGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, they expressed receptors for LDL. Importantly, when grown on collagen gels, hTERT-BME cells underwent MMP-dependent tube-like structure formation in response to VEGFR-2 activation. In a collagen gel sandwich assay, hTERT-BME formed tubular structures in the absence of exogenously added angiogenic cytokines. Sustained tube formation was induced by VEGF-A alone or in combination with FGF-2. From 17 sub-clones that displayed a non-transformed phenotype, a high proliferative capacity and tubulogenic properties in three-dimensional collagen gels, we isolated two distinct subpopulations that display a highly specific response to VEGF-A or to FGF-2. We have generated hTERT-BME cells that maintain endothelial-specific properties and function and have isolated clones that respond differentially to VEGF-A or FGF-2. These immortalized cell lines will facilitate the study of endothelial cell biology.

Tumour necrosis factor alpha confers an invasive, transformed phenotype on mammary epithelial cells.

Although loss of cell-cell adhesion and gain of invasive properties play a crucial role in the malignant progression of epithelial tumours, the molecular signals that trigger these processes have not been fully elucidated. In light of the well-established relationship between chronic inflammation and cancer, we hypothesized that pro-inflammatory cytokines disrupt epithelial-cell adhesion and promote cell migration. To test this hypothesis, we used an in vitro model in which 31EG4-2A4 mouse mammary epithelial cells grown in a collagen gel form compact spheroidal colonies. Among the several cytokines examined, tumour necrosis factor alpha (TNF-alpha) caused a pronounced 3D scattering of preformed epithelial-cell colonies and induced 31EG4-2A4 cells grown on top of a collagen gel to invade the underlying matrix. In addition, TNF-alpha abolished contact-mediated inhibition of cell proliferation and stimulated cell growth both in the absence of exogenous mitogens and under anchorage-independent conditions. TNF-alpha induced the expression of matrix metalloproteinase 9 (MMP-9). Addition of the MMP inhibitor BB-94 abrogated TNF-alpha-induced 3D scattering. TNF-alpha also enhanced the attachment of 31EG4-2A4 cells to type-I collagen and markedly increased the expression of the alpha2 integrin subunit. Addition of a blocking antibody to beta1-integrin or of rhodocetin (a specific alpha2beta1 antagonist) to collagen-gel cultures abrogated 3D scattering. Collectively, these results demonstrate an essential role for MMPs and alpha2beta1 integrin in the invasive response of 31EG4-2A4 cells to TNF-alpha. We propose that the biological activities described in this study contribute to the ability of TNF-alpha to promote tumour progression and cancer-cell dissemination.

Inducible expression of Snail selectively increases paracellular ion permeability and differentially modulates tight junction proteins.

Constitutive expression of the transcription factor Snail was previously shown to trigger complete epithelial-mesenchymal transition (EMT). The aim of this study was to determine whether inducible expression of Snail could modify epithelial properties without eliciting full mesenchymal conversion. For this purpose, we expressed mouse Snail (mSnail) cDNA in Madin-Darby canine kidney (MDCK) cells under the control of a doxycycline-repressible transactivator. Inducible expression of Snail did not result in overt EMT but induced a number of phenotypic alterations of MDCK cells, the most significant of which was the absence of fluid-filled blisterlike structures called "domes." To understand the mechanisms responsible for dome suppression, we assessed the effect of mSnail expression on epithelial barrier function. Although mSnail did not alter tight junction (TJ) organization and permeability to uncharged solutes, it markedly decreased transepithelial electrical resistance. In light of these findings, we evaluated the ability of MDCK cell monolayers to maintain ionic gradients and found that expression of mSnail selectively increases Na+ and Cl- permeability. Analysis of the expression of claudins, transmembrane proteins that regulate TJ ionic permeability, showed that mSnail induces a moderate decrease in claudin-2 and a substantial decrease in claudin-4 and -7 expression. Together, these results suggest that induction of mSnail selectively increases the ionic permeability of TJs by differentially modulating the expression of specific claudins.

Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells.

Overexpression of membrane-type-1 matrix metalloproteinase (MT1-MMP) in tumor cells has previously been shown to enhance tumor growth and metastasis. To establish if MT1-MMP is also able to confer tumorigenicity on nonmalignant epithelial cells, we transfected human MT1-MMP cDNA into Madin-Darby canine kidney (MDCK) cells expressing a tetracycline-repressible transactivator. Induction of MT1-MMP in the absence of doxycycline (Dox) was associated with activation of exogenous MMP-2 as well as with formation of large cysts and increased invasiveness in collagen matrices. Transfected cells were inoculated subcutaneously into two groups of nude mice, one of which received Dox to inhibit expression of MT1-MMP. Formation of tumor xenografts was observed in 11 of 17 mice maintained without Dox, but only in two of nine mice that received Dox (P<0.05). The xenografts were composed of tubular structures interspersed within a highly cellular stroma. The epithelial cells delimiting the lumen were polarized, as indicated by the basolateral distribution of Na,K-ATPase. Despite their differentiated appearance, the tumors lacked a well-defined boundary, and epithelial tubules invaded adjacent muscular layers. These results demonstrate that conditional expression of MT1-MMP in nonmalignant MDCK epithelial cells is by itself sufficient to drive formation of invasive tumors.

Generation and characterization of telomerase-transfected human lymphatic endothelial cells with an extended life span.

The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin alpha9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tube-like structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro.

Rapid transformation of white adipocytes into fat-oxidizing machines.

Adenovirus-induced hyperleptinemia rapidly depletes body fat in normal rats without increasing free fatty acids and ketogenesis, implying that fat-storing adipocytes are oxidizing the fat. To analyze the ultrastructural changes of adipocytes accompanying this functional transformation, we examined the fat tissue by electron microscopy. After 14 days of hyperleptinemia, adipocytes had become shrunken, fatless, and encased in a thick basement-membrane-like matrix. They were crowded with mitochondria that were much smaller than those of brown adipocytes. Their gene expression profile revealed striking up-regulation of peroxisome proliferator-activated receptor gamma coactivator 1alpha (an up-regulator of mitochondrial biogenesis not normally expressed in white fat), increased uncoupling proteins-1 and -2, and down-regulation of lipogenic enzymes. Phosphorylation of both acetyl CoA carboxylase and AMP-activated protein kinase was increased, thus explaining the increase in fatty acid oxidation. The ability to transform adipocytes into unique fat-burning cells may suggest novel therapeutic strategies for obesity.

Loss of active MEK1-ERK1/2 restores epithelial phenotype and morphogenesis in transdifferentiated MDCK cells.

Constitutive activation of the MAPK/ERK kinase (MEK)1-ERK2 signaling module in Madin-Darby canine kidney (MDCK)-C7 cells disrupts their ability to form cyst-like structures in collagen gels and induces an invasive, myofibroblast-like phenotype. However, the reversibility of these cellular events, as well as the relative role of both MEK isoforms (MEK1 and MEK2) and both ERK isoforms (ERK1 and ERK2) during these processes, has not yet been investigated. We now report that loss of constitutively active MEK1 (caMEK1) and, thus, loss of active ERK1/2 in C7caMEK1 cells is associated with increased MEK2 protein expression, reexpression of ERK1 protein, and epithelial redifferentiation of these cells. The morphological changes toward an epithelial phenotype in these revertant cell lines (C7rev4, C7rev5, C7rev7) are reflected by the upregulation of epithelial marker proteins, such as E-cadherin, beta-catenin, and cytokeratin, by the loss of alpha-smooth muscle actin expression, and by the ability of these epithelial revertants to form well-organized spherical cysts when grown in three-dimensional collagen gels. Further evidence for a role of the MEK1-ERK1/2 module in epithelial-mesenchymal transition was obtained from the analysis of two novel, spontaneously transdifferentiated MDCK-C7 cell clones (C7e1 and C7e2 cells). In these clones, increased MEK1/2-ERK1/2 phosphorylation, reduced MEK2 protein expression, and loss of ERK1 protein expression is associated with phenotypic alterations similar to those observed in transdifferentiated C7caMEK1 cells. C7e1 cells at least partially regained some of their epithelial characteristics at higher passages. In contrast, C7e2 cells maintained a transdifferentiated phenotype at high passage, were unable to generate cyst-like epithelial structures, and retained invasive properties when grown on a three-dimensional collagen matrix. We conclude that in renal epithelial MDCK-C7 cells, stable epithelial-to-mesenchymal transition (EMT) is associated with loss of ERK1 protein expression, reduced MEK2 protein expression, and increased basal ERK2 phosphorylation. In contrast, loss of active MEK1-ERK1/2 results in increased MEK2 protein expression and reexpression of ERK1 protein, concomitant with the restoration of epithelial phenotype and the ability to form cystic structures.