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.

CCL20 is up-regulated in non-alcoholic fatty liver disease fibrosis and is produced by hepatic stellate cells in response to fatty acid loading.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a prevalent complication of extreme obesity. Loading of the liver with fat can progress to inflammation and fibrosis including cirrhosis. The molecular factors involved in the progression from simple steatosis to fibrosis remain poorly understood. METHODS: Gene expression profiling using microarray, PCR array, and RNA sequencing was performed on RNA from liver biopsy tissue from patients with extreme obesity. Patients were grouped based on histological findings including normal liver histology with no steatosis, lobular inflammation, or fibrosis, and grades 1, 2, 3, and 4 fibrosis with coexistent steatosis and lobular inflammation. Validation of expression was conducted using quantitative PCR. Serum analysis was performed using ELISA. Expression analysis of hepatocytes and hepatic stellate cells in response to lipid loading were conducted in vitro using quantitative PCR and ELISA. RESULTS: Three orthogonal methods to profile human liver biopsy RNA each identified the chemokine CCL20 (CC chemokine ligand 20 or MIP-3 alpha) gene as one of the most up-regulated transcripts in NAFLD fibrosis relative to normal histology, validated in a replication group. CCL20 protein levels in serum measured in 224 NAFLD patients were increased in severe fibrosis (p < 0.001), with moderate correlation of hepatic transcript levels and serum levels. Expression of CCL20, but not its cognate receptor CC chemokine receptor 6, was significantly (p < 0.001) increased in response to fatty acid loading in LX-2 hepatic stellate cells, with relative increases greater than those in HepG2 hepatocyte cells. CONCLUSIONS: These results suggest that expression of CCL20, an important inflammatory mediator, is increased in NAFLD fibrosis. CCL20 serves as a chemoattractant molecule for immature dendritic cells, which have been shown to produce many of the inflammatory molecules that mediate liver fibrosis. These data also point to hepatic stellate cells as a key cell type that may respond to lipid loading of the liver.

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.

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.

Genetic Variants Implicate Dual Oxidase-2 in Familial and Sporadic Nonmedullary Thyroid Cancer.

Highly penetrant hereditary thyroid cancer manifests as familial nonmedullary thyroid cancer (FNMTC), whereas low-penetrance hereditary thyroid cancer manifests as sporadic disease and is associated with common polymorphisms, including rs965513[A]. Whole-exome sequencing of an FNMTC kindred identified a novel Y1203H germline dual oxidase-2 (DUOX2) mutation. DUOX2Y1203H is enzymatically active, with increased production of reactive oxygen species. Furthermore, patients with sporadic thyroid cancer homozygous for rs965513[A] demonstrated higher DUOX2 expression than heterozygous rs965513[A/G] or homozygous rs965513[A]-negative patients. These data suggest that dysregulated hydrogen peroxide metabolism is a common mechanism by which high- and low-penetrance genetic factors increase thyroid cancer risk. SIGNIFICANCE: This study provides novel insights into the genetic and molecular mechanisms underlying familial and sporadic thyroid cancers.

Bile acids upregulate BRCA1 and downregulate estrogen receptor 1 gene expression in ovarian cancer cells.

Two major risk factors for ovarian cancer include loss-of-function mutations in the BRCA1 (breast cancer 1, early onset) gene and aspects of estrogen metabolism. Modulation of the levels of the normal BRCA1 allele and estrogen receptor expression may therefore be a preventive strategy. Consensus binding motifs for the bile acid-responsive transcription factor farnesoid X receptor were identified in the BRCA1 and estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) genes, supported by chromatin immunoprecipitation sequencing data. Two major bile acids, deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), resulted in a greater than four-fold induction of BRCA1 transcript levels at 10 µmol/l and a greater than six-fold induction at 50 µmol/l relative to untreated control OVCAR3 ovarian cancer cells. Conversely, CDCA and DCA at 10 µmol/l resulted in about a 75% decrease in ESR1 expression in response to 10 µmol/l CDCA and DCA and close to 90% reduction with 50 µmol/l CDCA and DCA. Bile acids had no effects on ESR2 gene transcript levels. The inverse regulation of BRCA1 and ESR1 gene expression in response to physiological levels of bile acids could have important implications for disease penetrance and chemoprevention strategies in carriers of BRCA1 mutations.

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.

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.

The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum.

BACKGROUND: The increasing use of next generation DNA sequencing in clinical medicine is exposing the need for more genetics education in physician training. We piloted an initiative to determine the feasibility of incorporating exome sequencing data generated from DNA obtained from cadavers used for teaching Anatomy into a first year medical student integrated block-style course. METHODS: We optimized the procedure to obtain DNA for exome sequencing by comparing the quality and quantity of DNA isolated from several tissues by two different extraction methods. DNA was sequenced using exome capture and analyzed using standard methods. Single nucleotide variants (SNVs), as well as small insertions/deletions, with potential functional impact were selected by faculty for student teams to independently investigate and prepare presentations on their findings. RESULTS: A total of seven cadaver DNAs were sequenced yielding high quality results. SNVs were identified that were associated, with known physical traits and disease susceptibility, as well as pharmacogenomic phenotypes. Students presented findings based on correlation with known clinical information about the cadavers' diseases and traits. CONCLUSION: Exome sequencing of cadaver DNA is a useful tool to integrate Anatomy with Genetics and Biochemistry into a first year medical student core curriculum.

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.