Albumin endocytosis in endothelial cells induces TGF-beta receptor II signaling

Vascular endothelial cells undergo albumin endocytosis using a set of albumin binding proteins. This process is important for maintaining cellular homeostasis. We showed by several criteria that the previously described 73-kDa endothelial cell surface albumin binding protein is the 75-kDa transforming growth factor (TGF)-beta receptor type II (TbetaRII). Albumin coimmunoprecipitated with TbetaRII from a membrane fraction from rat lung microvascular endothelial cells. Albumin endocytosis-negative COS-7 cells became albumin endocytosis competent when transfected with wild-type TbetaRII but not when transfected with a domain-negative kinase mutant of TbetaRII. An antibody specific for TbetaRII inhibited albumin endocytosis. A mink lung epithelial cell line, which expresses both the TGF-beta receptor type I (TbetaRI) and the TbetaRII receptor, exhibited albumin binding to the cell surface and endocytosis. In contrast, mutant L-17 and DR-26 cells lacking TbetaRI or TbetaRII, respectively, each showed a dramatic reduction in binding and endocytosis. Albumin endocytosis induced Smad2 phosphorylation and Smad4 translocation as well as increased protein expression of the inhibitory Smad, Smad7. We identified regions of significant homology between amino acid sequences of albumin and TGF-beta, suggesting a structural basis for the interaction of albumin with the TGF-beta receptors and subsequent activation of TbetaRII signaling. The observed albumin-induced internalization of TbetaRII signaling may be an important mechanism in the vessel wall for controlling TGF-beta responses in endothelial cells.     

Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover

Endocytosis of cell surface receptors is an important regulatory event in signal transduction. The transforming growth factor beta (TGF-beta) superfamily signals to the Smad pathway through heteromeric Ser-Thr kinase receptors that are rapidly internalized and then downregulated in a ubiquitin-dependent manner. Here we demonstrate that TGF-beta receptors internalize into both caveolin- and EEA1-positive vesicles and reside in both lipid raft and non-raft membrane domains. Clathrin-dependent internalization into the EEA1-positive endosome, where the Smad2 anchor SARA is enriched, promotes TGF-beta signalling. In contrast, the lipid raft-caveolar internalization pathway contains the Smad7-Smurf2 bound receptor and is required for rapid receptor turnover. Thus, segregation of TGF-beta receptors into distinct endocytic compartments regulates Smad activation and receptor turnover.     

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.     

FKBP12 is a negative regulator of transforming growth factor-beta receptor internalization

Transforming growth factor-beta (TGF-beta) family polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGF-beta type I and II receptors. Although interaction screens have shown that the immunophilin FKBP12 interacts with TGF-beta type I receptors, the role of FKBP12 in TGF-beta receptor action is presently unclear. Using a chimeric TGF-beta receptor system, we have shown a specific enhancement of internalization when FKBP12 binding to the type I receptor was prevented with rapamycin. Moreover, although earlier studies demonstrated that type II receptor kinase activity was required for optimal internalization in mesenchymal cells, we found that rapamycin functioned downstream of the type II receptor kinase. Thus, rather than modulating TGF-beta signaling, our data suggest a novel role for FKBP12 as a negative regulator of TGF-beta receptor endocytosis.     

Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway

Members of the transforming growth factor β (TGF-β) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-β signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-β receptor (TGF-βR) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-βR internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-β-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-βR complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-βR endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.     

Betaglycan inhibits TGF-beta signaling by preventing type I-type II receptor complex formation. Glycosaminoglycan modifications alter betaglycan function

Transforming growth factor (TGF)-beta is a multifunctional growth factor with important roles in development, cell proliferation, and matrix deposition. It signals through the sequential activation of two serine/threonine kinase receptors, the type I and type II receptors. A third cell surface receptor, betaglycan, serves as a co-receptor for TGF-beta in some cell types, enhancing TGF-beta-mediated signaling. We have examined the function of betaglycan in renal epithelial LLC-PK1 cells that lack endogenous betaglycan. We demonstrate that the expression of betaglycan in LLC-PK1 cells results in inhibition of TGF-beta signaling as measured by reporter gene expression, thymidine incorporation, collagen production, and phosphorylation of the downstream signaling effectors Smad2 and Smad3. In comparison, the expression of betaglycan in L6 myoblasts enhances TGF-beta signaling, which is consistent with the published literature. The effects of betaglycan in LLC-PK1 cells are not mediated by ligand sequestration or increased production of a soluble form of the receptor, which has been reported to serve as a ligand antagonist. We demonstrate instead that in LLC-PK1 cells, unlike L6 cells, expression of betaglycan prevents association between the type I and type II TGF-beta receptors, which is required for signaling. This is a function of the glycosaminoglycan modifications of betaglycan. Betaglycan in LLC-PK1 cells exhibits higher molecular weight glycosaminoglycan (GAG) chains than in L6 cells, and a GAG- betaglycan mutant does not inhibit TGF-beta signaling or type I/type II receptor association in LLC-PK1 cells. Our data indicate that betaglycan can function as a potent inhibitor of TGF-beta signaling by a novel mechanism and provide support for an essential but complex role for proteoglycan co-receptors in growth factor signaling.     

Autocrine transforming growth factor-beta signaling mediates Smad-independent motility in human cancer cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor that plays a critical role in modulating cell growth, differentiation, and plasticity. There is increasing evidence that after cells lose their sensitivity to TGF-beta-mediated growth inhibition, autocrine TGF-beta signaling may potentially promote tumor cell motility and invasiveness. To understand the molecular mechanisms by which autocrine TGF-beta may selectively contribute to tumor cell motility, we have generated MDA-MB-231 breast cancer cells stably expressing a kinase-inactive type II TGF-beta receptor (T beta RII-K277R). Our data indicate that T beta RII-K277R is expressed, can associate with the type I TGF-beta receptor, and block both Smad-dependent and -independent signaling pathways activated by TGF-beta. In addition, wound closure and transwell migration assays indicated that the basal migratory potential of T beta RII-K277R expressing cells was impaired. The impaired motility of T beta RII-K277R cells could be restored by reconstituting TGF-beta signaling with a constitutively active TGF-beta type I receptor (ALK5(TD)) but not by reconstituting Smad signaling with Smad2/4 or Smad3/4 expression. In addition, the levels of ALK5(TD) expression sufficient to restore motility in the cells expressing T beta RII-K277R were associated with an increase in phosphorylation of Akt and extracellular signal-regulated kinase 1/2 but not Smad2. These data indicate that different signaling pathways require different thresholds of TGF-beta activation and suggest that TGF-beta promotes motility through mechanisms independent of Smad signaling, possibly involving activation of the phosphatidylinositol 3-kinase/Akt and/or mitogen-activated protein kinase pathways.     

Evidence that Smad2 is a tumor suppressor implicated in the control of cellular invasion.

The Smad2 protein plays an essential role in the transforming growth factor-beta (TGF-beta) signaling pathway. This pathway mediates growth inhibitory signals from the cell surface to the nucleus. Although Smad2 protein is significantly mutated in human cancers, there is no definitive evidence implicating Smad2 as a tumor-suppressor gene. Here we show that overexpression of the tumor-derived missense mutation Smad2.D450E, an unphosphorylable form of Smad2 found in colorectal and lung cancers, did not abolish the TGF-beta-mediated growth arrest, suggesting that resistance to the growth-inhibiting effects of TGF-beta exhibited by human tumors cannot be linked to the inactivation of Smad2 protein. In contrast, overexpression of Smad2.D450E induces cellular invasion, and this effect was enhanced by TGF-beta. A similar invasive phenotype was obtained in cells expressing another inactivating mutation in Smad2 (Smad2.P445H) found in colorectal cancer. These findings indicate that genetic defects in Smad2 are sufficient to confer the invasion-promoting effect of TGF-beta and reveal that TGF-beta acts through Smad2 to induce cellular invasion by a novel mechanism that is independent of Smad2 phosphorylation by the activated TGF-beta type I receptor.     

X-linked inhibitor of apoptosis protein functions as a cofactor in transforming growth factor-beta signaling

X-linked inhibitor of apoptosis protein (XIAP) is a potent suppressor of apoptotic cell death, which functions by directly inhibiting caspases, the principal effectors of apoptosis. Here we report that XIAP can also function as a cofactor in the regulation of gene expression by transforming growth factor-beta (TGF-beta). XIAP, but not the related proteins c-IAP1 or c-IAP2, associated with several members of the type I class of the TGF-beta receptor superfamily and potentiated TGF-beta-induced signaling. Although XIAP-mediated activation of c-Jun N-terminal kinase and nuclear factor kappa B was found to require the TGF-beta signaling intermediate Smad4, the ability of XIAP to suppress apoptosis was found to be Smad4-independent. These data implicate a role for XIAP in TGF-beta-mediated signaling that is distinct from its anti-apoptotic functions.     

Hyaluronan regulates transforming growth factor-beta1 receptor compartmentalization

Transforming growth factor-beta1 (TGF-beta1) is a key cytokine involved in the pathogenesis of fibrosis in many organs. We previously demonstrated in renal proximal tubular cells that the engagement of the extracellular polysaccharide hyaluronan with its receptor CD44 attenuated TGF-beta1 signaling. In the current study we examined the potential mechanism by which the interaction between hyaluronan (HA) and CD44 regulates TGF-beta receptor function. Affinity labeling of TGF-beta receptors demonstrated that in the unstimulated cells the majority of the receptor partitioned into EEA-1-associated non-lipid raft-associated membrane pools. In the presence of exogenous HA, the majority of the receptors partitioned into caveolin-1 lipid raft-associated pools. TGF-beta1 increased the association of activated/phosphorylated Smad proteins with EEA-1, consistent with activation of TGF-beta1 signaling following endosomal internalization. Following addition of HA, caveolin-1 associated with the inhibitory Smad protein Smad7, consistent with the raft pools mediating receptor turnover, which was facilitated by HA. Antagonism of TGF-beta1-dependent Smad signaling and the effect of HA on TGF-beta receptor associations were inhibited by depletion of membrane cholesterol using nystatin and augmented by inhibition of endocytosis. The effect of HA on TGF-beta receptor trafficking was inhibited by inhibition of HA-CD44 interactions, using blocking antibody to CD44 or inhibition of MAP kinase activation. In conclusion, we have proposed a model by which HA engagement of CD44 leads to MAP kinase-dependent increased trafficking of TGF-beta receptors to lipid raft-associated pools, which facilitates increased receptor turnover and attenuation of TGF-beta1-dependent alteration in proximal tubular cell function.     

Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors

The generation of mice lacking specific components of the transforming growth factor-beta (TGF-beta) signal tranduction pathway shows that TGF-beta is a key player in the development and physiology of the cardiovascular system. Both pro- and anti-angiogenic properties have been ascribed to TGF-beta, for which the molecular mechanisms are unclear. Here we report that TGF-beta can activate two distinct type I receptor/Smad signalling pathways with opposite effects. TGF-beta induces phosphorylation of Smad1/5 and Smad2 in endothelial cells and these effects can be blocked upon selective inhibition of ALK1 or ALK5 expression, respectively. Whereas the TGF-beta/ALK5 pathway leads to inhibition of cell migration and proliferation, the TGF-beta/ALK1 pathway induces endothelial cell migration and proliferation. We identified genes that are induced specifically by TGF-beta-mediated ALK1 or ALK5 activation. Id1 was found to mediate the TGF-beta/ALK1-induced (and Smad-dependent) migration, while induction of plasminogen activator inhibitor-1 by activated ALK5 may contribute to the TGF-beta-induced maturation of blood vessels. Our results suggest that TGF-beta regulates the activation state of the endothelium via a fine balance between ALK5 and ALK1 signalling.