km23-1/DYNLRB1 regulation of MEK/ERK signaling and R-Ras in invasive human colorectal cancer cells.

We previously found that km23-1/DYNLRB1 is required for transforming growth factor-ß (TGFß) production through Ras/ERK pathways in TGFß-sensitive epithelial cells and in human colorectal cancer (CRC) cells. Here we demonstrate that km23-1/DYNLRB1 is required for mitogen-activated protein kinase kinase (MEK) activation in human CRC cells, detected by km23-1/DYNLRB1-siRNA inhibition of phospho-(p)-MEK immunostaining in RKO cells. Furthermore, we show that CRISPR-Cas9 knock-out (KO) of km23-1/DYNLRB1 reduced cell migration in two additional CRC models, HCT116 and DLD-1. Of interest, in contrast to our previous work showing that dynein motor activity was required for TGFß-mediated nuclear translocation of Smad2, in the current report, we demonstrate for the first time that disruption of dynein motor activity did not reduce TGFß-mediated activation of MEK1/2 or c-Jun N-terminal kinase (JNK). Moreover, size exclusion chromatography of RKO cell lysates revealed that B-Raf, extracellular signal-regulated kinase (ERK), and p-ERK were not present in the large molecular weight fractions containing dynein holocomplex components. Furthermore, sucrose gradient fractionation of cell lysates from both HCT116 and CBS CRC cells demonstrated that km23-1/DYNLRB1 co-sedimented with Ras, p-ERK, and ERK in fractions that did not contain components of holo-dynein. Thus, km23-1/DYNLRB1 may be associated with activated Ras/ERK signaling complexes in cell compartments that do not contain the dynein holoprotein complex, suggesting dynein-independent km23-1/DYNLRB1 functions in Ras/ERK signaling. Finally, of the Ras isoforms, R-Ras is most often associated with cell migration, adhesion, and protrusive activity. Here, we show that a significant fraction of km23-1/DYNLRB1 and RRas wase co-localized at the protruding edges of migrating HCT116 cells, suggesting an important role for the km23-1/DYNLRB1-R-Ras complex in CRC invasion.

Requirement for protein kinase A in the phosphorylation of the TGFß receptor-interacting protein km23-1 as a component of TGFß downstream effects.

km23-1 was previously identified as a TGFß-receptor interacting protein that was phosphorylated on serines after TGFß stimulation. In the current report, we examined the role of km23-1 phosphorylation in the downstream effects of TGFß/protein kinase A (PKA) signaling. Using phosphorylation site prediction software, we found that km23-1 has two potential PKA consensus phosphorylation sites. In vitro kinase assays further demonstrated that PKA directly phosphorylates km23-1 on serine 73 (S73). Moreover, our results show that the PKA-specific inhibitor H89 diminishes phosphorylation of km23-1 on S73 after TGFß stimulation. Taken together, our results demonstrate that TGFß induction of PKA activity results in phosphorylation of km23-1 on S73. In order to assess the mechanisms underlying PKA phosphorylation of km23-1 on S73 (S73-km23-1) after TGFß stimulation, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGFß regulates complex formation between the PKA regulatory subunit RIß and km23-1 in vivo. In addition, an S73A mutant of km23-1 (S73A-km23-1), which could not be phosphorylated by PKA, inhibited TGFß induction of the km23-1-dynein complex and transcriptional activation of the activin-responsive element (ARE). Furthermore, our results show that km23-1 is required for cAMP-responsive element (CRE) transcriptional activation by TGFß, with S73-km23-1 being required for the CRE-dependent TGFß stimulation of fibronectin (FN) transcription. Collectively, our results demonstrate for the first time that TGFß/PKA phosphorylation of km23-1 on S73 is required for ARE- and CRE-mediated downstream events that include FN induction.

The TGFß receptor-interacting protein km23-1/DYNLRB1 plays an adaptor role in TGFß1 autoinduction via its association with Ras.

We have previously elucidated the signaling events that are required for TGFß1 autoinduction (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765-30773). Further, we have reported that the TGFß receptor (TßR)-interacting protein km23-1 plays an important role in TGFß signal transduction (Jin, Q., Ding, W., and Mulder, K. M. (2007) J. Biol. Chem. 282, 19122-19132). Here we examined the role of km23-1 in TGFß1 autoinduction in TGFß-sensitive epithelial cells. siRNA blockade of km23-1 reduced TGFß1 mRNA expression, as well as DNA binding and transcriptional activation of the relevant activator protein-1 site in the human TGFß1 promoter. Further, knockdown of km23-1 inhibited TGFß-mediated activation of ERK and JNK, phosphorylation of c-Jun, and transactivation of the c-Jun promoter. Sucrose gradient analyses indicate that km23-1 was present in lipid rafts together with Ras and TßRII after TGFß treatment. Immunoprecipitation/blot analyses revealed the formation of a TGFß-inducible complex between Ras and km23-1 in vivo within minutes of TGFß addition. Moreover, we demonstrate for the first time that km23-1 is required for Ras activation by TGFß. Our results indicate that km23-1 is required for TGFß1 autoinduction through Smad2-independent Ras/ERK/JNK pathways. More importantly, our findings demonstrate that km23-1 functions as a critical adaptor coupling TßR activation to activation of Ras effector pathways downstream.

Knockdown of c-Fos suppresses the growth of human colon carcinoma cells in athymic mice.

Here we have investigated whether inhibition of c-Fos expression in RKO human colon carcinoma cells (HCCCs) would result in reduced TGFß1 expression and suppression of tumor growth in athymic mice. We stably transfected RKO cells with c-Fos small interfering RNA (siRNA) or with the corresponding control siRNA. Using these stable cell lines, we demonstrated that siRNA-c-Fos significantly suppressed both AP-1 binding, promoter reporter activity at the proximal AP-1 site in the TGFß1 promoter, and TGFß1 production. Further, we established colon cancer xenografts with each of RKO-siRNA-EV, RKO-siRNA-Ctrl and RKO-siRNA-c-Fos cells. By 24 days, the tumor size of RKO-siRNA-c-Fos xenografts was 40% that of either RKO-EV or RKO-siRNA-Ctrl. Immunohistochemistry (IHC) of tumor xenografts demonstrated that siRNA-c-Fos significantly blocked c-Fos expression, and consequently expression of TGFß1. However, expression of TGFß2 and TGFß3 were unaffected. Overall, our results demonstrate that blockade of TGFß1 production by siRNA-c-Fos effectively suppressed tumor growth in vivo.

Role of km23-1 in RhoA/actin-based cell migration.

km23-1 was originally identified as a TGFß receptor-interacting protein that plays an important role in TGFß signaling. Moreover, km23-1 is actually part of an ancient superfamily of NTPase-regulatory proteins, widely represented in archaea and bacteria. To further elucidate the function of km23-1, we identified novel protein interacting partners for km23-1 by using tandem affinity purification (TAP) and tandem mass spectrometry (MS). Here we show that km23-1 interacted with a class of proteins involved in actin-based cell motility and modulation of the actin cytoskeleton. We further showed that km23-1 modulates the formation of a highly organized stress fiber network. More significantly, we demonstrated that knockdown (KD) of km23-1 decreased RhoA activation in Mv1Lu epithelial cells. Finally, our results demonstrated for the first time that depletion of km23-1 inhibited cell migration of human colon carcinoma cells (HCCCs) in wound-healing assays. Overall, our findings demonstrate that km23-1 regulates RhoA and motility-associated actin modulating proteins, suggesting that km23-1 may represent a novel target for anti-metastatic therapy.

Overexpression of the dynein light chain km23-1 in human ovarian carcinoma cells inhibits tumor formation in vivo and causes mitotic delay at prometaphase/metaphase.

km23-1 is a dynein light chain that was identified as a TGFß receptor-interacting protein. To investigate whether km23-1 controls human ovarian carcinoma cell (HOCC) growth, we established a tet-off inducible expression system in SKOV-3 cells in which the expression of km23-1 is induced upon doxycycline removal. We found that forced expression of km23-1 inhibited both anchorage-dependent and anchorage-independent growth of SKOV-3 cells. More importantly, induction of km23-1 expression substantially reduced the tumorigenicity of SKOV-3 cells in a xenograft model in vivo. Fluorescence-activated cell sorting analysis of SKOV-3 and IGROV-1 HOCCs demonstrated that the cells were accumulating at G2/M. Phospho-MEK, phospho-ERK and cyclin B1 were elevated, as was the mitotic index, suggesting that km23-1 suppresses HOCCs growth by inducing a mitotic delay. Immunofluorescence analyses demonstrated that the cells were accumulating at prometaphase/metaphase with increases in multipolar and multinucleated cells. Further, although the mitotic spindle assembly checkpoint protein BubR1 was present at the prometaphase kinetochore in Dox+/- cells, it was inappropriately retained at the metaphase kinetochore in Dox- cells. Thus, the mechanism by which high levels of km23-1 suppress ovarian carcinoma growth in vitro and inhibit ovary tumor formation in vivo appears to involve a BubR1-related mitotic delay.

Requirement of a dynein light chain in TGFbeta/Smad3 signaling.

We have previously reported that the dynein light chain (DLC) km23-1 is required for Smad2-dependent TGFbeta signaling. Here we describe another member of the km23/DYNLRB/LC7/robl family of DLCs, termed km23-2, which is also involved in TGFbeta signaling. We show not only that TGFbeta stimulates the interaction of km23-2 (DYNLRB2) with TGFbeta receptor II (TbetaRII) but also that TGFbeta regulates the interaction between km23-2 and endogenous TbetaRII in vivo. In addition, TGFbeta treatment causes km23-2 phosphorylation, whereas a kinase-deficient form of TbetaRII prevents km23-2 phosphorylation. In contrast to the km23-1 isoform, blockade of km23-2 expression using small interfering RNAs (siRNAs) decreased key TGFbeta/Smad3-specific responses, including the induction of both plasminogen activator inhibitor-1 (PAI-1) gene expression and p21 protein expression. Blockade of km23-1 expression had no effect on these two major TGFbeta/Smad3 responses under similar conditions. Further, km23-2 was required for TGFbeta stimulation of Smad3-dependent Smad-binding element (SBE)2-Luc transcriptional activity, but not for TGFbeta stimulation of Smad2-dependent activin responsive element (ARE)-Lux transcriptional activity. In order to assess the mechanisms underlying the preferential stimulation of Smad3- versus Smad2-specific TGFbeta responses, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGFbeta stimulated preferential complex formation of km23-2 with Smad3, relative to Smad2. Collectively, our findings indicate that km23-2 is required for Smad3-dependent TGFbeta signaling. More importantly, we demonstrate that km23-2 has functions in TGFbeta signaling that are distinct from those for km23-1. This is the first report to describe a differential requirement for unique isoforms of a specific DLC family in Smad-specific TGFbeta signaling.

A novel transforming growth factor-beta receptor-interacting protein that is also a light chain of the motor protein dynein.

The phosphorylated, activated cytoplasmic domains of the transforming growth factor-beta (TGFbeta) receptors were used as probes to screen an expression library that was prepared from a highly TGFbeta-responsive intestinal epithelial cell line. One of the TGFbeta receptor-interacting proteins isolated was identified to be the mammalian homologue of the LC7 family (mLC7) of dynein light chains (DLCs). This 11-kDa cytoplasmic protein interacts with the TGFbeta receptor complex intracellularly and is phosphorylated on serine residues after ligand-receptor engagement. Forced expression of mLC7-1 induces specific TGFbeta responses, including an activation of Jun N-terminal kinase (JNK), a phosphorylation of c-Jun, and an inhibition of cell growth. Furthermore, TGFbeta induces the recruitment of mLC7-1 to the intermediate chain of dynein. A kinase-deficient form of TGFbeta RII prevents both mLC7-1 phosphorylation and interaction with the dynein intermediate chain (DIC). This is the first demonstration of a link between cytoplasmic dynein and a natural growth inhibitory cytokine. Furthermore, our results suggest that TGFbeta pathway components may use a motor protein light chain as a receptor for the recruitment and transport of specific cargo along microtublules.

A transforming growth factor-beta receptor-interacting protein frequently mutated in human ovarian cancer.

Ovarian carcinomas, particularly recurrent forms, are frequently resistant to transforming growth factor-beta (TGF-beta)-mediated growth inhibition. However, mutations in the TGF-beta receptor I and receptor II (TbetaR-I and TbetaR-II) genes have only been reported in a minority of ovarian carcinomas, suggesting that alterations in TGF-beta-signaling components may play an important role in the loss of TGF-beta responsiveness. Using laser-capture microdissection and nested reverse-transcription-PCR, we found that km23, which interacts with the TGF-beta receptor complex, is altered at a high frequency in human ovarian cancer patients. A novel form of km23, missing exon 3 (Deltaexon3-km23), was found in 2 of 19 tumor tissues from patients with ovarian cancer. In addition to this alteration, a stop codon mutation (TAA --> CAC) was detected in two patients. This alteration results in an elongated protein, encoding 107-amino-acid residues (Delta107km23), instead of the wild-type 96-amino-acid form of km23. Furthermore, five missense mutations (T38I, S55G, T56S, I89V, and V90A) were detected in four patients, providing a total alteration rate of 42.1% (8 of 19 cases) in ovarian cancer. No km23 alterations were detected in 15 normal tissues. Such a high alteration rate in ovarian cancer suggests that km23 may play an important role in either TGF-beta resistance or tumor progression in this disease. In keeping with these findings, the functional studies described herein indicate that both the Deltaexon3-km23 and S55G/I89V-km23 mutants displayed a disruption in binding to the dynein intermediate chain in vivo, suggesting a defect in cargo recruitment to the dynein motor complex. In addition, the Deltaexon3-km23 resulted in an inhibition of TGF-beta-dependent transcriptional activation of both the p3TP-lux and activin responsive element reporters. Collectively, our results suggest that km23 alterations found in ovarian cancer patients result in altered dynein motor complex formation and/or aberrant transcriptional regulation by TGF-beta.

Structure and dynamics of the homodimeric dynein light chain km23.

km23 (96 residues, 11 kDa) is the mammalian ortholog of Drosophila roadblock, the founding member of LC7/robl/km23 class of dynein light chains. km23 has been shown to be serine-phosphorylated following TGFbeta receptor activation and to bind the dynein intermediate chain in response to such phosphorylation. Here, we report the three-dimensional solution structure of km23, which is shown to be that of a homodimer, similar to that observed for the heterodimeric complex formed between p14 and MP1, two distantly related members of the MglB/robl superfamily, but distinct from the LC8 and Tctex-1 classes of dynein light chains, which also adopt homodimeric structures. The conserved surface residues of km23, including three serine residues, are located predominantly on a single face of the molecule. Adjacent to this face is a large cleft formed by the incomplete overlap of loops from opposite monomers. As shown by NMR relaxation data collected at two fields, several cleft residues are flexible on the ns-ps and ms-mus timescales. Based on these observations, we propose that the patch of conserved residues on the central face of the molecule corresponds to the site at which km23 binds the dynein intermediate chain and that the flexible cleft formed between the overlap of loops from the two monomers corresponds to the site at which km23 binds other partners, such as the TGFbeta type II receptor or Smad2.

Requirement of km23 for TGFbeta-mediated growth inhibition and induction of fibronectin expression.

We previously identified km23 as a novel TGFbeta receptor-interacting protein. Here we show that km23 is ubiquitously expressed in human tissues and that cell-type specific differences in endogenous km23 protein expression exist. In addition, we demonstrate that the phosphorylation of km23 is TGFbeta-dependent, in that EGF was unable to phosphorylate km23. Further, the kinase activity of both TGFbeta receptors appears to play a role in the TGFbeta-mediated phosphorylation of km23, although TGFbeta RII kinase activity is absolutely required for km23 phosphorylation. Blockade of km23 using small interfering RNAs significantly decreased key TGFbeta responses, including induction of fibronectin expression and inhibition of cell growth. Thus, our results demonstrate that km23 is required for TGFbeta induction of fibronectin expression and is necessary, but not sufficient, for TGFbeta-mediated growth inhibition.

A dynein motor attachment complex regulates TGFß/Smad3 signaling.

Our previous results have demonstrated that km23-2 has functions in TGFß signaling that are distinct from those for km23-1. In the current report, we demonstrate that blockade of km23-2 decreased TGFß activation of the human Smad7 promoter Smad7-Luc, an endogenous Smad3-target promoter. Luminescence-based mammalian interaction mapping (LUMIER) analyses showed that TGFß stimulated the interaction of km23-2 preferentially with Smad3, relative to that with Smad2. Size exclusion chromatography experiments revealed that km23-2 and Smad3 were recruited into the same complex after TGFß treatment. Moreover, in the presence of TGFß, but not in the absence, km23-2 was present in early endosomes with the TGFß receptors (TßRs) and Smad3. Collectively, our data indicate that km23-2 is a critical signaling intermediate in a Smad3-dependent TGFß signaling pathway. We also provide evidence of the novel finding that TGFß stimulates the rapid recruitment of the km23-2 dimer to the dynein intermediate chain (DIC) of the dynein complex, whereas a kinase-deficient form of TßRII prevented this interaction. Finally, we demonstrate for the first time that TGFß stimulated not only assembly of the dynein motor attachment complex, but also triggered the tethering of the km23-2-Smad3 cargo to the other dynein components. Thus, our data demonstrate a novel function for km23-2 as a motor receptor to recruit Smad3 to the dynein complex for intracellular transport, thereby mediating Smad3-dependent TGFß signaling.

Decreased tumor progression and invasion by a novel anti-cell motility target for human colorectal carcinoma cells.

We have previously described a novel modulator of the actin cytoskeleton that also regulates Ras and mitogen-activated protein kinase activities in TGFß-sensitive epithelial cells. Here we examined the functional role of this signaling regulatory protein (km23-1) in mediating the migration, invasion, and tumor growth of human colorectal carcinoma (CRC) cells. We show that small interfering RNA (siRNA) depletion of km23-1 in human CRC cells inhibited constitutive extracellular signal-regulated kinase (ERK) activation, as well as pro-invasive ERK effector functions that include phosphorylation of Elk-1, constitutive regulation of c-Fos-DNA binding, TGFß1 promoter transactivation, and TGFß1 secretion. In addition, knockdown of km23-1 reduced the paracrine effects of CRC cell-secreted factors in conditioned medium and in fibroblast co-cultures. Moreover, km23-1 depletion in human CRC cells reduced cell migration and invasion, as well as expression of the ERK-regulated, metastasis-associated scaffold protein Ezrin. Finally, km23-1 inhibition significantly suppressed tumor formation in vivo. Thus, our results implicate km23-1 as a novel anti-metastasis target for human colon carcinoma cells, capable of decreasing tumor growth and invasion via a mechanism involving suppression of various pro-migratory features of CRC. These include a reduction in ERK signaling, diminished TGFß1 production, decreased expression of the plasma membrane-cytoskeletal linker Ezrin, as well as attenuation of the paracrine effects of colon carcinoma-secreted factors on fibroblast migration and mitogenesis. As such, km23-1 inhibitors may represent a viable therapeutic strategy for interfering with colon cancer progression and invasion.

Requirement of a dynein light chain in transforming growth factor ß signaling in zebrafish ovarian follicle cells.

We have previously reported that the dynein light chains km23-1 and km23-2 are required for TGFß signaling in mammalian cells. Here we describe another member of the km23/DYNLRB/LC7/robl family of dynein light chains in zebrafish, termed zkm23, which is also involved in TGFß signaling. zkm23 was rapidly phosphorylated after TGFß stimulation. TGFß RII kinase activity was absolutely required for zkm23 phosphorylation, whereas a constitutively active TGFß RI did not induce phosphorylation. Further, TGFß stimulated a rapid recruitment of the zkm23 dynein light chain to the dynein intermediate chain of the dynein complex, and the TGFß RII kinase was required for this interaction. Finally, blockade of zkm23 using morpholino oligos resulted in an inhibition of TGFß-mediated transcriptional responses. Thus, our results demonstrate for the first time that the dynein light chain zkm23 is required for TGFß signaling in cultured zebrafish ovarian follicle cells.

Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-beta signaling pathway.

We have identified km23-1 as a novel transforming growth factor-beta (TGFbeta) receptor (TbetaR)-interacting protein that is also a light chain of the motor protein dynein (dynein light chain). Herein, we demonstrate by sucrose gradient analyses that, in the presence of TGFbeta but not in the absence, km23-1 was present in early endosomes with the TbetaRs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGFbeta treatment, prior to Smad2 translocation to the nucleus. In addition, immunoprecipitation/blot analyses showed that TGFbeta regulated the interaction between endogenous km23-1 and endogenous Smad2 in vivo. Blockade of km23-1 using a small interfering RNA approach resulted in a reduction in both total intracellular Smad2 levels and in nuclear levels of phosphorylated Smad2 after TGFbeta treatment. This decrease was reversed by lactacystin, a specific inhibitor of the 26 S proteasome, suggesting that knockdown of km23-1 causes proteasomal degradation of phosphorylated (i.e. activated) Smad2. Blockade of km23-1 also resulted in a reduction in TGFbeta/Smad2-dependent ARE-Lux transcriptional activity, which was rescued by a km23-1 small interfering RNA-resistant construct. In contrast, a reduction in TGFbeta/Smad3-dependent SBE2-Luc transcriptional activity did not occur under similar conditions. Furthermore, overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocked TGFbeta-stimulated nuclear translocation of Smad2. Collectively, our findings indicate for the first time that a dynein light chain is required for a Smad2-dependent TGFbeta signaling pathway.

Requirement of Smad3 and CREB-1 in mediating transforming growth factor-beta (TGF beta) induction of TGF beta 3 secretion.

Because increased transforming growth factor-beta (TGFbeta) production by tumor cells contributes to cancer progression through paracrine mechanisms, identification of critical points that can be targeted to block TGFbeta production is important. Previous studies have identified the precise signaling components and promoter elements required for TGFbeta induction of TGFbeta1 expression in epithelial cells (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765-30773). To determine how regulation of TGFbeta3 expression differs from that of TGFbeta1, we identified the precise signaling pathways and transcription factor-binding sites that are required for TGFbeta3 gene expression. By using mutational analysis in electrophoresis mobility shift assays (EMSAs), we demonstrated that the c-AMP-responsive element (CRE) site in the TGFbeta3 promoter was required for TGFbeta-inducible TGFbeta3 expression. Electrophoresis mobility supershift assays indicated that CRE-binding protein 1 (CREB1) and Smad3 were the major components present in this TGFbeta-inducible complex. Furthermore, by using chromatin immunoprecipitation assays, we demonstrated that CREB-1, ATF-2, and c-Jun bound constitutively at the TGFbeta3 promoter (-100 to +1), whereas Smad3 bound at this site only after TGFbeta stimulation. In addition, inhibition of JNK and p38 suppressed TGFbeta induction of TGFbeta3 transactivation, whereas inhibition of ERK and protein kinase A had no effect. Small interfering RNA-CREB1 and small interfering RNA-Smad3 significantly inhibited TGFbeta stimulation of TGFbeta3 promoter reporter activity and TGFbeta3 production. Our results indicate that TGFbeta activation of the TGFbeta3 promoter CRE site, which leads to TGFbeta3 production, is required for TGFbetaRII, JNK, p38, and Smad3 but was independent of protein kinase A, ERK, and Smad4.

c-Fos is required for TGFbeta1 production and the associated paracrine migratory effects of human colon carcinoma cells.

In tumor cells that have lost responsiveness to the growth inhibitory effects of transforming growth factor beta (TGFbeta), increased TGFbeta production by the tumor cells often contributes to cancer progression, primarily through paracrine mechanisms. Here we investigated the major components of the activator protein-1 (AP-1) complex in the TGFbeta1 promoter of human colon carcinoma cells (HCCCs). In contrast to untransformed epithelial cells (UECs), HCCCs displayed constitutive activation of AP-1 at the proximal AP-1 site in the human TGFbeta1 promoter. Further, in contrast to the JunD and Fra-2 components present in the AP-1 complex at this AP-1 site in UECs, c-Fos was the major detectable AP-1 component in HCCCs. Thus, transcriptional factor switching had occurred in HCCCs relative to the UECs, with regard to the proximal AP-1 site of the human TGFbeta1 promoter. Small interfering RNAs (siRNAs) against c-Fos significantly suppressed AP-1 activity at the relevant AP-1 site, and led to a decrease in TGFbeta1 secretion by the HCCCs. Our results indicate for the first time that c-Fos binding at the TGFbeta1 promoter proximal AP-1 site in HCCCs is required for TGFbeta1 production by the tumor cells. Further, we demonstrated that blockade of TGFbeta1 secretion by c-Fos siRNA led to a suppression of the cellular migration and mitogenesis of NIH 3T3 fibroblasts in a paracrine fashion. Thus, c-Fos may have utility as a target for blocking tumor cell-secreted TGFbeta1, thereby suppressing the migratory behavior associated with the malignant phenotype of HCCCs.

Requirement of TGF-beta receptor-dependent activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases (Sapks) for TGF-beta up-regulation of the urokinase-type plasminogen activator receptor.

We have previously demonstrated that activation of the Ras/Mapk pathways is required for transforming growth factor beta (TGF-beta) induction of TGF-beta(1) expression. Here we examined the role of the Ras/Mapk pathways in TGF-beta induction of urokinase-type plasminogen activator receptor (uPAR) expression in untransformed intestinal epithelial cells (IECs). TGF-beta activated the stress-activated protein kinases (Sapk)/c-Jun N-terminal kinases (JNKs) within 5-10 min, an effect that preceeded TGF-beta induction of uPAR expression in these cells. TGF-beta induction of both JNK1 activity and JunD phosphorylation was blocked by expression of a dominant-negative mutant of the type II TGF-beta receptor (DN TbetaRII), a dominant-negative mutant of MKK4 (DN MKK4), or a dominant-negative mutant of Ras (RasN17), or by the addition of the JNK inhibitor SP600125. TGF-beta also induced AP-1 complex formation at the distal AP-1 site (-184 to -178) of the uPAR promoter within 2 h of TGF-beta addition, consistent with the time-dependent up-regulation of uPAR expression. The primary components present in the TGF-beta-stimulated AP-1 complex bound to the uPAR promoter were Jun D and Fra-2. Moreover, addition of SP600125, or expression of DN MKK4 or DN TbetaRII, blocked TGF-beta up-regulation of uPAR in IECs. Accordingly, our results indicate that TGF-beta activates the Ras/MKK4/JNK1 signaling cascade, leading to induction of AP-1 activity, which, in turn, up-regulates uPAR expression. Our results also indicate that the type II TGF-beta receptor (RII) is required for TGF-beta activation of JNK1 and the resulting up-regulation of uPAR expression.