Philip H. Howe

TGF-Beta Induces Fibronectin Synthesis Through a c-Jun N-terminal Kinase-Dependent, Smad4-Independent Pathway

Transforming growth factor‐β (TGF‐β) exerts its effects on cell proliferation, differentiation and migration in part through its modulation of extracellular matrix components, such as fibronectin and plasminogen activator inhibitor‐1 (PAI‐1). Although the SMAD family of proteins recently has been shown to be a key participant in TGF‐β signaling, other signaling pathways have also been shown to be activated by TGF‐β. We report here that c‐Jun N‐terminal kinase (JNK), a member of the MAP kinase family, is activated in response to TGF‐β in the human fibrosarcoma HT1080‐derived cell line BAHgpt. Stable expression of dominant‐negative forms of JNK1 and MKK4, an upstream activator of JNK, results in loss of TGF‐β‐stimulated fibronectin mRNA and protein induction, while having little effect on TGF‐β‐induced levels of PAI‐1. The human fibronectin promoter contains three CRE elements, one of which has been shown to bind a c‐Jun–ATF‐2 heterodimer. Utilizing a GAL4 fusion trans‐reporting system, we demonstrate a decrease in transactivating potential of GAL4–c‐Jun and GAL4–ATF‐2 in dominant‐negative JNK1‐ and MKK4‐expressing cells. Finally, we show that TGF‐β‐induced fibronectin synthesis is independent of Smad4. These results demonstrate that TGF‐β‐mediated fibronectin induction requires activation of JNK which in turn modulates the activity of c‐Jun and ATF‐2 in a Smad4independent manner.

The adaptor molecule Disabled-2 links the transforming growth factor β receptors to the Smad pathway

Using a genetic complementation approach we have identified disabled‐2 (Dab2), a structural homolog of the Dab1 adaptor molecule, as a critical link between the transforming growth factor β (TGFβ) receptors and the Smad family of proteins. Expression of wild‐type Dab2 in a TGFβ‐signaling mutant restores TGFβ‐mediated Smad2 phosphorylation, Smad translocation to the nucleus and Smad‐dependent transcriptional responses. TGFβ stimulation triggers a transient increase in association of Dab2 with Smad2 and Smad3, which is mediated by a direct interaction between the N‐terminal phosphotyrosine binding domain of Dab2 and the MH2 domain of Smad2. Dab2 associates with both the type I and type II TGFβ receptors in vivo, suggesting that Dab2 is part of a multiprotein signaling complex. Together, these data indicate that Dab2 is an essential component of the TGFβ signaling pathway, aiding in transmission of TGFβ signaling from the TGFβ receptors to the Smad family of transcriptional activators.

Evidence That Ser87 of BimEL Is Phosphorylated by Akt and Regulates BimEL Apoptotic Function

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of BimEL, one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of BimEL correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked BimEL phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-BimEL fusion protein and identified the Akt phosphorylation site in the BimEL domain as Ser87. Further, we demonstrated that cytokine stimulation promotes BimEL binding to 14-3-3 proteins. Finally, we show that mutation of Ser87 dramatically increases the apoptotic potency of BimEL. We propose that Ser87 of BimEL is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of BimEL, thereby promoting cell survival.

TGF-beta-mediated phosphorylation of hnRNP E1 induces EMT via transcript-selective translational induction of Dab2 and ILEI.

Transforming growth factor-β (TGF-β) induces epithelial–mesenchymal transdifferentiation (EMT) accompanied by cellular differentiation and migration. Despite extensive transcriptomic profiling, the identification of TGF-β-inducible, EMT-specific genes has met with limited success. Here we identify a post-transcriptional pathway by which TGF-β modulates the expression of EMT-specific proteins and of EMT itself. We show that heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) binds a structural, 33-nucleotide TGF-β-activated translation (BAT) element in the 3′ untranslated region of disabled-2 (Dab2) and interleukin-like EMT inducer (ILEI) transcripts, and represses their translation. TGF-β activation leads to phosphorylation at Ser 43 of hnRNP E1 by protein kinase Bβ/Akt2, inducing its release from the BAT element and translational activation of Dab2 and ILEI messenger RNAs. Modulation of hnRNP E1 expression or its post-translational modification alters the TGF-β-mediated reversal of translational silencing of the target transcripts and EMT. These results suggest the existence of a TGF-β-inducible post-transcriptional regulon that controls EMT during the development and metastatic progression of tumours.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: Focus on hnRNP E1's multifunctional regulatory roles

Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a family of RNA-binding proteins. The complexity and diversity associated with the hnRNPs render them multifunctional, involved not only in processing heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs, but also acting as trans-factors in regulating gene expression. Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1), a subgroup of hnRNPs, is a KH-triple repeat containing RNA-binding protein. It is encoded by an intronless gene arising from hnRNP E2 through a retrotransposition event. hnRNP E1 is ubiquitously expressed and functions in regulating major steps of gene expression, including pre-mRNA processing, mRNA stability, and translation. Given its wide-ranging functions in the nucleus and cytoplasm and interaction with multiple proteins, we propose a post-transcriptional regulon model that explains hnRNP E1s widespread functional diversity.

Oxidized Low Density Lipoprotein and Lysophosphatidylcholine Stimulate Cell Cycle Entry in Vascular Smooth Muscle Cells EVIDENCE FOR RELEASE OF FIBROBLAST GROWTH FACTOR-2

We have previously shown that oxidized low density lipoprotein (LDL) but not native LDL stimulated DNA synthesis in cultured smooth muscle cells (SMC) and that α-tocopherol (vitamin E) inhibited this proliferative response (Lafont, A., Chai, Y. C., Cornhill, J. F., Whitlow, P. L., Howe, P. H., and Chisolm, G. M. (1995) J. Clin. Invest. 95, 1018-1025). The moiety of oxidized LDL that stimulates DNA synthesis and the cellular mechanism for this potentially mitogenic effect are not known. We now report that lipid fractions containing lysophospholipids from oxidized LDL or phospholipase A2-treated native LDL stimulated SMC DNA synthesis as did palmitoyl lysophosphatidylcholine (lysoPC). Protein kinase C inhibitors and down-regulation of protein kinase C activity by phorbol ester inhibited oxidized LDL- and lysoPC-induced DNA synthesis. A neutralizing monoclonal antibody against fibroblast growth factor-2 significantly inhibited oxidized LDL and lysoPC-induced DNA synthesis in SMC; irrelevant antibodies were ineffective. Vitamin E inhibited the DNA synthesis stimulated by lysoPC, an observation that distinguished this effect from DNA synthesis induced by another detergent, digitonin. These results suggest that oxidized LDL and its lysoPC moiety stimulate SMC to enter the cell cycle via an oxidative mechanism that causes the release of fibroblast growth factor-2 and a subsequent autocrine or paracrine response.

Regulation of the Wnt signaling pathway by disabled-2 (Dab2)

The adaptor molecule Disabled‐2 (Dab2) has been shown to link cell surface receptors to downstream signaling pathways. Using a small‐pool cDNA screening strategy, we identify that the N‐terminal domain of Dab2 interacts with Dishevelled‐3 (Dvl‐3), a signaling mediator of the Wnt pathway. Ectopic expression of Dab2 in NIH‐3T3 mouse fibroblasts attenuates canonical Wnt/β‐catenin‐mediated signaling, including accumulation of β‐catenin, activation of β‐catenin/T‐cell‐specific factor/lymphoid enhancer‐binding factor 1‐dependent reporter constructs, and endogenous cyclin D1 induction. Wnt stimulation leads to a time‐dependent dissociation of endogenous Dab2–Dvl‐3 and Dvl‐3–axin interactions in NIH‐3T3 cells, while Dab2 overexpression leads to maintenance of Dab2–Dvl‐3 association and subsequent loss of Dvl‐3–axin interactions. In addition, we find that Dab2 can associate with axin in vitro and stabilize axin expression in vivo. Mouse embryo fibroblasts which lack Dab2 exhibit constitutive Wnt signaling as evidenced by increased levels of nuclear β‐catenin and cyclin D1 protein levels. Based on these results, we propose that Dab2 functions as a negative regulator of canonical Wnt signaling by stabilizing the β‐catenin degradation complex, which may contribute to its proposed role as a tumor suppressor.

Effect of alpha-tocopherol on restenosis after angioplasty in a model of experimental atherosclerosis.

The ability of alpha-tocopherol to reduce restenosis after angioplasty was tested in a rabbit model in which angioplasty was performed on established atherosclerotic lesions. Lesions induced by 4 wk of cholesterol feeding after focal desiccation of femoral arteries were balloon dilated. 3 wk after angioplasty, angiographically determined minimum luminal diameters were less in the untreated group (0.80 +/- 0.51 mm) than in the group treated with oral alpha-tocopherol beginning 19 d before angioplasty (1.38 +/- 0.29 mm; P < 0.01). The cross-sectional area of the intima-media was greater in the untreated group (1.18 +/- 0.48 mm2) than in the alpha-tocopherol group (0.62 +/- 0.25 mm2, P < 0.0001). These differences were not due to vasoconstriction or altered plasma cholesterol. Alpha-tocopherol thus reduced restenosis after angioplasty in this model. In rabbit vascular smooth muscle cells, oxidized low density lipoprotein stimulated DNA synthesis. Alpha-tocopherol treatment inhibited DNA synthesis stimulated by oxidized low density lipoprotein, but not by serum. The findings are consistent with the hypothesis that oxidized lipids can stimulate hyperplasia and that antioxidants may limit hyperplasia by inhibiting either the oxidation or the proliferative effects of oxidants on cells.

Smad3 Potentiates Transforming Growth Factor β (TGFβ)-induced Apoptosis and Expression of the BH3-only Protein Bim in WEHI 231 B Lymphocytes

Transforming growth factor-β (TGFβ) is a potent growth inhibitor and inducer of apoptosis in B lymphocytes and is essential for immune regulation and maintenance of self-tolerance. Here we show that exogenous overexpression of Smad3 potentiates TGFβ-induced apoptosis and expression of the pro-apoptotic protein Bim in WEHI 231 B lymphocytes. Overexpression of dominant-negative forms of Smad3 abrogate these TGFβ-induced responses. We also demonstrate that TGFβ induces Bim protein expression concomitant with its induction of apoptosis in the mouse progenitor B lymphocyte cell line, Ba/F3. Enhanced expression of Bim protein induced by TGFβ is associated with an increased association of Bim with Bcl-2 and a concomitant loss of mitochondrial membrane potential. Furthermore, we find that the anti-apoptotic effect of the pro-survival cytokine CD40 results in the abrogation of TGFβ-mediated Bim induction. Our data provide the first evidence of Bim expression levels that are increased by the addition of a pro-apoptotic cytokine, TGFβ, and also suggest that the TGFβ−specific transcription factor Smad3 plays a role in mediating Bim expression levels and apoptosis.

Type II Transforming Growth Factor-β Receptor Recycling Is Dependent upon the Clathrin Adaptor Protein Dab2

Transforming growth factor-β receptor recycling is regulated by the clathrin adaptor Dab2 protein. In the absence of Dab2, receptors localize in a perinuclear locale because they are unable to transit from the early endosomal antigen 1-positive early endosome to the Rab11-positive endosomal recycling compartment.