Smad7 differentially regulates transforming growth factor beta-mediated signaling pathways

Smad7 has been identified as a negative regulator of transforming growth factor beta (TGF-beta) signaling by interfering with the phosphorylation of other Smad proteins by TGF-beta receptor type I (TbetaRI). We established a mink lung epithelial (Mv1Lu) cell line where ectopic expression of Smad7 is tightly controlled by doxycycline using an improved Tet-on system. Once induced by doxycycline, the recombinant Smad7 was localized predominantly in the perinuclear region and in the cytoplasm. However, the type of culture surface alters the subcellular localization of Smad7: on plastic or on fibronectin-coated glass, Smad7 was localized in the cytoplasm; but when the cells were cultured on glass, nuclear localization was observed. TGF-beta stimulation did not alter substantially the cellular distribution of Smad7. Importantly, the expression of recombinant Smad7 differentially inhibited TGF-beta signaling pathways. Consistent with previous studies, Smad7 inhibited TGF-beta-stimulated induction of type 1 plasminogen activator inhibitor as measured by p3TP-Lux reporter. However, expression of Smad7 had little effect on TGF-beta-induced growth inhibition.     

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation

Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.     

A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A

Transforming growth factor beta (TGFbeta) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFbeta signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFbeta activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFbeta activation of CREB, induction of p21(Cip1), and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFbeta and PKA signaling pathways.     

Calcium signals in growth factor signal transduction

There is a substantial amount of information which has been obtained concerning the effects of growth factors on [Ca²⁺]_i in proliferating cells. A number of different mitogens are known to induce elevations in [Ca²⁺]_i and some characterization of the Ca²⁺ response to different classes of mitogens has been obtained. In addition, much is known about whether the Ca²⁺ response to a particular growth factor occurs as the result of an influx of external Ca²⁺ or a mobilization of internal Ca²⁺ stores. In addition, a considerable amount of information is available on the mechanism by which the Ins(1,4,5)P₃-sensitive internal Ca²⁺ store takes up and releases Ca²⁺. However, there is still a large deficiency in our information concerning other Ca²⁺ stores in proliferating cells as well as in our knowledge of the mechanisms for regulating Ca²⁺ entry pathways. Much more data addressing these issues exists for other types of agonist-stimulated cells, and we have discussed much of it in this review article. While the wealth of data in nonproliferating cells provides some indications of what mechanisms might be involved in the growth factor-induced changes in [Ca²⁺]_i, it is clear that much work must be done in proliferating cells to fully understand how external factors such as growth factors control [Ca²⁺]_i. In addition, much work remains to be done in identifying the mechanisms for the internal control of [Ca²⁺]_i as cells move through the cell cycle and in identifying the role that these changes in [Ca²⁺]_i may play throughout the cell cycle.     

Cell-type-specific activation of PAK2 by transforming growth factor beta independent of Smad2 and Smad3

Transforming growth factor beta (TGF-beta) causes growth arrest in epithelial cells and proliferation and morphological transformation in fibroblasts. Despite the ability of TGF-beta to induce various cellular phenotypes, few discernible differences in TGF-beta signaling between cell types have been reported, with the only well-characterized pathway (the Smad cascade) seemingly under identical control. We determined that TGF-beta receptor signaling activates the STE20 homolog PAK2 in mammalian cells. PAK2 activation occurs in fibroblast but not epithelial cell cultures and is independent of Smad2 and/or Smad3. Furthermore, we show that TGF-beta-stimulated PAK2 activity is regulated by Rac1 and Cdc42 and dominant negative PAK2 or morpholino antisense oligonucleotides to PAK2 prevent the morphological alteration observed following TGF-beta addition. Thus, PAK2 represents a novel Smad-independent pathway that differentiates TGF-beta signaling in fibroblast (growth-stimulated) and epithelial cell (growth-inhibited) cultures.     

Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation

Renal microsomal cytochrome P-450 monooxygenase-dependent metabolism of arachidonic acid generates a series of regioisomeric epoxyeicosatrienoic acids that can be further metabolized by soluble epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acids. Evidence exists that these metabolites affect renal function and, in particular, blood pressure regulation. To examine this possibility, blood pressure and renal arachidonic acid metabolism were examined in mice with a targeted disruption of the soluble epoxide hydrolase gene. Systolic blood pressure of male soluble epoxide hydrolase-null mice was lower compared with wild-type mice in both the absence and presence of dietary salt loading. Both female soluble epoxide hydrolase-null and wild-type female mice also had significantly lower systolic blood pressure than male wild-type mice. Renal formation of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids was markedly lower for soluble epoxide hydrolase-null versus wild-type mice of both sexes. Although disruption of soluble epoxide hydrolase in female mice had minimal effects on blood pressure, deletion of this gene feminized male mice by lowering systolic blood pressure and altering arachidonic acid metabolism. These data provide the first direct evidence for a role for soluble epoxide hydrolase in blood pressure regulation and identify this enzyme as a novel and attractive target for therapeutic intervention in hypertension.     

Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads

Upon transforming growth factor beta (TGF-beta) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. We find that TGF-beta-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-beta-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-beta-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties.     

Endoglin promotes transforming growth factor beta-mediated Smad 1/5/8 signaling and inhibits endothelial cell migration through its association with GIPC

Transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation, migration, and angiogenesis, the ALK-1/Smad 1/5/8 and ALK-5/Smad2/3 pathways. Endoglin is a co-receptor predominantly expressed in endothelial cells that participates in TGFbeta-mediated signaling with ALK-1 and ALK-5 and regulates critical aspects of cellular and biological responses. The embryonic lethal phenotype of knock-out mice because of defects in angiogenesis and disease-causing mutations resulting in human vascular diseases both support essential roles for endoglin, ALK-1, and ALK-5 in the vasculature. However, the mechanism by which endoglin mediates TGF-beta signaling through ALK-1 and ALK-5 has remained elusive. Here we describe a novel interaction between endoglin and GIPC, a scaffolding protein known to regulate cell surface receptor expression and trafficking. Co-immunoprecipitation and immunofluorescence confocal studies both demonstrate a specific interaction between endoglin and GIPC in endothelial cells, mediated by a class I PDZ binding motif in the cytoplasmic domain of endoglin. Subcellular distribution studies demonstrate that endoglin recruits GIPC to the plasma membrane and co-localizes with GIPC in a TGFbeta-independent manner, with GIPC-promoting cell surface retention of endoglin. Endoglin specifically enhanced TGF-beta1-induced phosphorylation of Smad 1/5/8, increased a Smad 1/5/8 responsive promoter, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with GIPC. These studies define a novel mechanism for the regulation of endoglin signaling and function in endothelial cells and demonstrate a new role for GIPC in TGF-beta signaling.     

The intracellular form of notch blocks transforming growth factor beta-mediated growth arrest in Mv1Lu epithelial cells

Notch signaling influences a variety of cell fate decisions during development, and constitutive activation of the pathway can provoke unbridled cell growth and cancer. The mechanisms by which Notch affects cell growth are not well established. We describe here a novel link between Notch and cell cycle control. We found that Mv1Lu epithelial cells harboring an oncogenic form of Notch (NICD) are resistant to the cell cycle-inhibitory effects of transforming growth factor beta (TGF-beta). NICD did not affect TGF-beta signaling per se but blocked induction of the Cdk inhibitor p15(INK4B). c-Myc, whose down-regulation by TGF-beta is required for p15(INK4B) induction, remained elevated in the NICD-expressing cells. c-Myc expression was also maintained in low serum, indicating that Notch's effects on c-Myc are not specific to TGF-beta. Our results are consistent with a model in which a strong Notch signal indirectly deregulates c-Myc expression and thereby renders Mv1Lu epithelial cells resistant to growth-inhibitory signals.     

Integrin-linked kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition

The role of integrin-linked kinase (ILK) in transforming growth factor beta (TGFbeta)-mediated epithelial to mesenchymal transition was investigated. A stable transfection of dominant-negative ILK results in the prevention of TGFbeta-mediated E-cadherin delocalization. TGFbeta-mediated phosphorylation of Akt at Ser-473 was inhibited by dominant-negative ILK and PI3K inhibitors, LY294002 and wortmannin. Treatment with TGFbeta stimulated induction of Akt and ILK kinase activity in HaCat control cells. This increased ILK activity by TGFbeta was lowered by PI3K inhibitor, LY294002. In addition, PI3K inhibitor, dominant-negative Akt, and dominant-negative ILK could not block TGFbeta-mediated C-terminal phosphorylation of Smad2. Taken together, these data suggest that PI3K-ILK-Akt pathway that is independent of the TGFbeta-induced Smad pathway is required for TGFbeta-mediated epithelial to mesenchymal transition.