Identification of disease-associated microRNA in a diet-induced model of nonalcoholic steatohepatitis.

Emerging evidence suggests that microRNA dysregulation plays an important role in nonalcoholic steatohepatitis. Using a model of diet-induced liver disease that progresses to fibrosis and hepatocellular carcinoma, we identify a set of 22 microRNA with robust correlation with liver enzyme levels and liver collagen content. These disease-asssociated miRs play pivotal roles in steatosis, extracellular matrix deposition and liver cancer, and may form the basis for identification of therapeutic strategies against this form of liver disease.

Collagen Characterization in a Model of Nonalcoholic Steatohepatitis with Fibrosis; A Call for Development of Targeted Therapeutics.

Left untreated, nonalcoholic fatty liver disease can progress to nonalcoholic steatohepatitis (NASH), fibrosis, and end-stage liver disease. To date, few if any therapies have proven effective against NASH with fibrosis. Quantification and qualification of hepatic scar might enable development of more effective targeted therapies. In a murine model of NASH induced by diet, we characterized fibrillar collagen deposition within the hepatic parenchyma. At harvest, livers from the modified diet cohort exhibited NASH with fibrosis. Transcriptomic analysis of hepatic tissue revealed increased col1a1, col1a2, and col3a1, each of which correlated directly with hepatic hydroxyproline content. Circular polarized microscopic analysis of Picrosirius red-stained liver sections revealed deposition of collagen type I within the parenchyma. Development of therapeutics designed to mitigate collagen type I accumulation might prove effective in NASH with fibrosis.

Renal Function Improvement Following ANG-3777 Treatment in Patients at High Risk for Delayed Graft Function After Kidney Transplantation.

BACKGROUND: Patients (20%-50%) undergoing renal transplantation experience acute kidney injury resulting in delayed graft function. ANG-3777 is an hepatocyte growth factor mimetic that binds to the c-MET receptor. In animal models, ANG-3777 decreases apoptosis, increases proliferation, and promotes organ repair and function. METHODS: This was a randomized, double-blind, placebo-controlled, phase 2 trial of patients undergoing renal transplantation with <50 cc/h urine output for 8 consecutive hours over the first 24 hours posttransplantation, or creatinine reduction ratio <30% from pretransplantation to 24 hours posttransplantation. Subjects were randomized as 2:1 to 3, once-daily IV infusions of ANG-3777, 2 mg/kg (n = 19), or placebo (n = 9). Primary endpoint: time in days to achieve ≥1200 cc urine for 24 hours. RESULTS: Patients treated with ANG-3777 were more likely to achieve the primary endpoint of 1200 cc urine for 24 hours by 28 days posttransplantation (83.3% versus 50% placebo; log-rank test: χ2 = 2.799, P = 0.09). Compared with placebo, patients in the ANG-3777 arm had larger increases in urine output; lower serum creatinine; greater reduction in C-reactive protein and neutrophil gelatinase-associated lipocalin; fewer dialysis sessions and shorter duration of dialysis; fewer hospital days; significantly less graft failure; and higher estimated glomerular filtration rate. Adverse events occurred in a similar percentage of subjects in both arms. Events per subject were twice as high in the placebo arm. CONCLUSIONS: There was an efficacy signal for improved renal function in subjects treated with ANG-3777 relative to placebo, with a good safety profile.

Liver Biopsy Hydroxyproline Content Is a Diagnostic for Hepatocellular Carcinoma in Murine Models of Nonalcoholic Steatohepatitis.

There is increasing evidence that nonalcoholic steatohepatitis (NASH) is a risk factor for hepatocellular carcinoma (HCC) in the absence of cirrhosis, a phenomenon termed noncirrhotic HCC. Early diagnosis of HCC is critical to a favorable prognosis. We tested the hypothesis that hydroxyproline content of liver biopsy samples is diagnostic for HCC in murine models of NASH induced by diet or by diet and chemicals. The training set comprised mice fed a standard diet or a fast-food diet with or without administration of thioacetamide. At harvest, livers from the modified diet cohort exhibited NASH with a subset of NASH livers exhibiting HCC. Hydroxyproline content was measured in liver biopsy samples with tissue in the NASH+HCC cohort sampled from the remote, nontumor parenchyma. Plotting the receiver operating characteristics (ROC) with hydroxyproline as the continuous variable against the absence or presence of HCC yielded an area under ROC of 0.87, a threshold of >0.18 µg hydroxyproline/mg liver and sensitivity of 91% with a specificity of 83.3%. The use of liver hydroxyproline content as a diagnostic for HCC in a test set comprising healthy, NASH and NASH+HCC livers proved 87% accurate.

Glycerol-3-phosphate Acyltransferase1 Is a Model-Agnostic Node in Nonalcoholic Fatty Liver Disease: Implications for Drug Development and Precision Medicine.

Left untreated nonalcoholic fatty liver disease (NAFLD) can progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. The observed failure of clinical trials in NASH may suggest that current model systems do not fully recapitulate human disease, and/or hallmark pathological features of NASH may not be driven by the same pathway in every animal model let alone in each patient. Identification of a model-agnostic disease-associated node can spur the development of effective drugs for the treatment of liver disease. Glycerol-3-phosphate acyltransferase1 (GPAT1) plays a pivotal role in lipid accumulation by shunting fats away from oxidation. In the present study, hepatic GPAT1 expression was evaluated in three etiologically different models of NAFLD. Compared to the sham cohort, hepatic GPAT1 mRNA levels were elevated by ∼5-fold in steatosis and NASH with fibrosis with immunofluorescent staining revealing increased GPAT1 in the fatty liver. A significant and direct correlation (r = 0.88) was observed between hepatic GPAT1 mRNA expression and severity of the liver disease. Picrosirius red staining revealed a logarithmic relation between hepatic GPAT1 mRNA expression and scar. These data suggest that hepatic GPAT1 is an early disease-associated model-agnostic node in NAFLD and form the basis for the development of a potentially successful therapeutic against NASH.

Collagen-driven remodeling of the intrahepatic duct wall in the PCK rat model of polycystic kidney disease-Caroli syndrome.

AIM OF THE STUDY: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation and expansion of cysts within the kidney. Caroli syndrome (CS) is characterized by cystic saccular dilatation of intrahepatic ducts. Kidney and liver images from a model of ADPKD-CS were evaluated to characterize remodeling of the cystically dilated intrahepatic duct wall and the renal cyst wall. MATERIAL AND METHODS: Archival digitized images from Masson's trichrome-stained renal and Picrosirius red (PSR)-stained renal and hepatic cross-sections were sourced from the PCK rat model of ADPKD-CS, and age-matched Sprague-Dawley rats (wild-type). Cross-sectional areas and wall thicknesses of renal cysts and intrahepatic ducts were measured. Circularly polarized PSR microscopy was utilized to observe accumulation of collagen and identify its subtype. RESULTS: In the PCK rat model of ADPKD-CS, renal cysts were relatively thin-walled in comparison to intrahepatic ducts with renal cyst cross-sectional area to wall ratio 47-fold greater than the corresponding ratio in intrahepatic ducts. Increasing intrahepatic duct cross-sectional area was accompanied by a rapid and steep rise in wall thickness. There was a weak but significant direct correlation (r = 0.49, p = 0.037) between renal cyst cross-sectional area and wall thickness. Circularly polarized Picrosirius red microscopy revealed collagen I accumulation within the walls of dilated intrahepatic ducts but not renal cysts. CONCLUSIONS: These data suggest that unlike renal cysts, cystically dilated intrahepatic ducts undergo collagen-driven wall remodeling in the PCK rat.

Remodeling of Intrahepatic Ducts in a Model of Caroli Syndrome: Is Scar Carcinoma a Consequence of Laplace's Law?

Caroli syndrome, characterized by saccular dilatation of intrahepatic ducts and congenital hepatic fibrosis, is without therapy in part due to its ultra-rare prevalence and the apparent lack of availability of a suitable experimental model. While the PCK rat has long been used as a model of fibropolycystic kidney disease, hepatobiliary biophysics in this animal model is incompletely characterized. Compared to age-matched, wild-type controls, the PCK rat demonstrated severe hepatomegaly and large saccular dilated intrahepatic ducts. Nevertheless, hepatic density was greater in the PCK rat, likely due to severe duct wall sclerosis accompanied by scarring across the hepatic parenchyma. Extracellular matrix accumulation appeared proportional to duct cross-sectional area and liver volume and appeared compensatory in nature. The PCK rat livers exhibited both cholangiocarcinoma and hepatocellular carcinoma coincident with areas of increased extracellular matrix deposition. Together, these data suggest that the PCK rat model mimics at least in part the spectrum of hepatobiliary pathology observed in Caroli syndrome and highlights the attendant risk associated with this disease.

A small molecule fibrokinase inhibitor in a model of fibropolycystic hepatorenal disease.

AIM: To evaluate the novel platelet-derived growth factor receptor and vascular endothelial growth factor receptor dual kinase inhibitor ANG3070 in a polycystic kidney disease-congenital hepatic fibrosis model. METHODS: At 6 wk of age, PCK rats were randomized to vehicle or ANG3070 for 4 wk. At 10 wk, 24 h urine and left kidneys were collected and rats were continued on treatment for 4 wk. At 14 wk, 24 h urine was collected, rats were sacrificed, and liver and right kidneys were collected for histological evaluation. For Western blot studies, PCK rats were treated with vehicle or ANG3070 for 7 d and sacrificed approximately 30 min after the last treatments. RESULTS: Compared to the wild-type cohort, the PCK kidney (Vehicle cohort) exhibited a marked increase in kidney and liver mass, hepato-renal cystic volume, hepato-renal fibrosis and hepato-renal injury biomarkers. Intervention with ANG3070 in PCK rats decreased kidney weight, reduced renal cystic volume and reduced total kidney hydroxyproline, indicating significantly reduced rental interstitial fibrosis compared to the PCK-Vehicle cohort. ANG3070 treatment also mitigated several markers of kidney injury, including urinary neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, cystatin C and interleukin-18 levels. In addition, this treatment attenuated key indices of renal dysfunction, including proteinuria, albuminuria and serum blood urea nitrogen and creatinine, and significantly improved renal function compared to the PCK-Vehicle cohort. ANG3070 treatment also significantly decreased liver enlargement, hepatic lesions, and liver fibrosis, and mitigated liver dysfunction compared to the PCK-Vehicle cohort. CONCLUSION: These results suggest that ANG3070 has the potential to slow disease, and may serve as a bridge toward hepato-renal transplantation in patients with fibropolycystic disease.

Deep Learning in Drug Discovery and Medicine; Scratching the Surface.

The practice of medicine is ever evolving. Diagnosing disease, which is often the first step in a cure, has seen a sea change from the discerning hands of the neighborhood physician to the use of sophisticated machines to use of information gleaned from biomarkers obtained by the most minimally invasive of means. The last 100 or so years have borne witness to the enormous success story of allopathy, a practice that found favor over earlier practices of medical purgatory and homeopathy. Nevertheless, failures of this approach coupled with the omics and bioinformatics revolution spurred precision medicine, a platform wherein the molecular profile of an individual patient drives the selection of therapy. Indeed, precision medicine-based therapies that first found their place in oncology are rapidly finding uses in autoimmune, renal and other diseases. More recently a new renaissance that is shaping everyday life is making its way into healthcare. Drug discovery and medicine that started with Ayurveda in India are now benefiting from an altogether different artificial intelligence (AI)-one which is automating the invention of new chemical entities and the mining of large databases in health-privacy-protected vaults. Indeed, disciplines as diverse as language, neurophysiology, chemistry, toxicology, biostatistics, medicine and computing have come together to harness algorithms based on transfer learning and recurrent neural networks to design novel drug candidates, a priori inform on their safety, metabolism and clearance, and engineer their delivery but only on demand, all the while cataloging and comparing omics signatures across traditionally classified diseases to enable basket treatment strategies. This review highlights inroads made and being made in directed-drug design and molecular therapy.

Supervised learning reveals circulating biomarker levels diagnostic of hepatocellular carcinoma in a clinically relevant model of non-alcoholic steatohepatitis; An OAD to NASH.

Although cirrhosis is a key risk factor for the development of hepatocellular carcinoma (HCC), mounting evidence indicates that in a subset of patients presenting with non-alcoholic steatohepatitis (NASH) HCC manifests in the absence of cirrhosis. Given the sheer size of the ongoing non-alcoholic fatty liver disease (NAFLD) epidemic and the dismal prognosis associated with late-stage primary liver cancer there is an urgent need for HCC surveillance in the NASH population. Using serum levels of HCC biomarkers as vectors and biopsy-proven HCC or no HCC as outputs / binary classifier, a supervised learning campaign was undertaken to develop a minimally invasive technique for making a diagnosis of HCC in a clinically relevant model of NASH. Adult mice randomized to control diet or a fast food diet (FFD) were followed for up to 14 mo and serum level of a panel of HCC-relevant biomarkers was compared with liver biopsies at 3 and 14 mo. Both NAFLD Activity Score (NAS) and hepatic hydroxyproline content were elevated at 3 and 14 mo on FFD. Picrosirius red staining of liver sections revealed a filigree pattern of fibrillar collagen deposition with no cirrhosis at 14 mo on FFD. Nevertheless, 46% of animals bore one or more tumors on their livers confirmed as HCC in hematoxylin-eosin-stained liver sections. In this training set, receiver operating characteristic (ROC) curves analysis for serum levels of the HCC biomarkers osteopontin (OPN), alpha-fetoprotein (AFP) and Dickkopf-1 (DKK1) returned concordance-statistic/area under ROC curve of ≥ 0.89. Serum levels of OPN (threshold, 218 ng/mL; sensitivity, 82%; specificity, 86%), AFP (136 ng/mL; 91%; 97%) and DKK1 (2.4 ng/mL; 82%; 81%) diagnostic for HCC were confirmed in a test set comprising mice on control diet or FFD and mice subjected to hepatic ischemia-reperfusion injury. These data suggest that levels of circulating OPN, AFP and DKK1 can be used to make a diagnosis of HCC in a clinically relevant model of NASH.

A modified elliptical formula to estimate kidney collagen content in a model of chronic kidney disease.

The extent of scarring or renal interstitial collagen deposition in chronic kidney disease (CKD) can only be ascertained by highly invasive, painful and sometimes risky, tissue biopsy. Interestingly, while CKD-related abnormalities in kidney size can often be visualized using ultrasound, not only does the ellipsoid formula used today underestimate true renal size, but the calculated renal size does not inform tubulointerstitial collagen content. We used coronal kidney sections from healthy mice and mice with kidney disease to develop a new formula for estimating renal parenchymal area. While treating the kidney as an ellipse with the major axis (a) the polar distance, this technique involves extending the minor axis (b) into the renal pelvis to obtain a new minor axis, be. The calculated renal parenchymal area is remarkably similar to the true or measured area. Biochemically determined kidney collagen content revealed a strong and positive correlation with the calculated renal parenchymal area. Picrosirius red staining for tubulointerstitial collagen also correlated with calculated renal parenchymal area. The extent of renal scarring, i.e. kidney interstitial collagen content, can now be computed by making just two axial measurements which can easily be accomplished via noninvasive imaging of this organ.

Anti-steatotic and anti-fibrotic effects of the KCa3.1 channel inhibitor, Senicapoc, in non-alcoholic liver disease.

AIM: To evaluate a calcium activated potassium channel (KCa3.1) inhibitor attenuates liver disease in models of non-alcoholic fatty liver disease (NAFLD). METHODS: We have performed a series of in vitro and in vivo studies using the KCa3.1 channel inhibitor, Senicapoc. Efficacy studies of Senicapoc were conducted in toxin-, thioacetamide (TAA) and high fat diet (HFD)-induced models of liver fibrosis in rats. Efficacy and pharmacodynamic effects of Senicapoc was determined through biomarkers of apoptosis, inflammation, steatosis and fibrosis. RESULTS: Upregulation of KCa3.1 expression was recorded in TAA-induced and high fat diet-induced liver disease. Treatment with Senicapoc decreased palmitic acid-driven HepG2 cell death. (P < 0.05 vs control) supporting the finding that Senicapoc reduces lipid-driven apoptosis in HepG2 cell cultures. In animals fed a HFD for 6 wk, co-treatment with Senicapoc, (1) reduced non-alcoholic fatty liver disease (NAFLD) activity score (NAS) (0-8 scale), (2) decreased steatosis and (3) decreased hepatic lipid content (Oil Red O, P < 0.05 vs vehicle). Randomization of TAA animals and HFD fed animals to Senicapoc was associated with a decrease in liver fibrosis as evidenced by hydroxyproline and Masson's trichrome staining (P < 0.05 vs vehicle). These results demonstrated that Senicapoc mitigates both steatosis and fibrosis in liver fibrosis models. CONCLUSION: These data suggest that Senicapoc interrupts more than one node in progressive fatty liver disease by its anti-steatotic and anti-fibrotic activities, serving as a double-edged therapeutic sword.

An Empirical Biomarker-Based Calculator for Cystic Index in a Model of Autosomal Recessive Polycystic Kidney Disease-The Nieto-Narayan Formula.

Autosomal recessive polycystic kidney disease (ARPKD) is associated with progressive enlargement of the kidneys fuelled by the formation and expansion of fluid-filled cysts. The disease is congenital and children that do not succumb to it during the neonatal period will, by age 10 years, more often than not, require nephrectomy+renal replacement therapy for management of both pain and renal insufficiency. Since increasing cystic index (CI; percent of kidney occupied by cysts) drives both renal expansion and organ dysfunction, management of these patients, including decisions such as elective nephrectomy and prioritization on the transplant waitlist, could clearly benefit from serial determination of CI. So also, clinical trials in ARPKD evaluating the efficacy of novel drug candidates could benefit from serial determination of CI. Although ultrasound is currently the imaging modality of choice for diagnosis of ARPKD, its utilization for assessing disease progression is highly limited. Magnetic resonance imaging or computed tomography, although more reliable for determination of CI, are expensive, time-consuming and somewhat impractical in the pediatric population. Using a well-established mammalian model of ARPKD, we undertook a big data-like analysis of minimally- or non-invasive blood and urine biomarkers of renal injury/dysfunction to derive a family of equations for estimating CI. We then applied a signal averaging protocol to distill these equations to a single empirical formula for calculation of CI. Such a formula will eventually find use in identifying and monitoring patients at high risk for progressing to end-stage renal disease and aid in the conduct of clinical trials.

Late intervention with the small molecule BB3 mitigates postischemic kidney injury.

Ischemia-reperfusion-mediated acute kidney injury can necessitate renal replacement therapy and is a major cause of morbidity and mortality. We have identified BB3, a small molecule, which when first administered at 24 h after renal ischemia in rats, improved survival, augmented urine output, and reduced the increase in serum creatinine and blood urea nitrogen. Compared with control kidneys, the kidneys of BB3-treated animals exhibited reduced levels of kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, and reduced tubular apoptosis and acute tubular necrosis but enhanced tubular regeneration. Consistent with its hepatocyte growth factor-like mode of action, BB3 treatment promoted phosphorylation of renal cMet and Akt and upregulated renal expression of the survival protein Bcl-2. These data suggest that the kidney is amenable to pharmacotherapy even 24 h after ischemia-reperfusion and that activation of the hepatocyte growth factor signaling pathway with the small molecule BB3 confers interventional benefits late into ischemia-reperfusion injury. These data formed, in part, the basis for the use of BB3 in a clinical trial in kidney recipients presenting with delayed graft function.

Sustained functional improvement by hepatocyte growth factor-like small molecule BB3 after focal cerebral ischemia in rats and mice.

Hepatocyte growth factor (HGF), efficacious in preclinical models of acute central nervous system injury, is burdened by administration of full-length proteins. A multiinstitutional consortium investigated the efficacy of BB3, a small molecule with HGF-like activity that crosses the blood-brain barrier in rodent focal ischemic stroke using Stroke Therapy Academic Industry Roundtable (STAIR) and Good Laboratory Practice guidelines. In rats, BB3, begun 6 hours after temporary middle cerebral artery occlusion (tMCAO) reperfusion, or permanent middle cerebral artery occlusion (pMCAO) onset, and continued for 14 days consistently improved long-term neurologic function independent of sex, age, or laboratory. BB3 had little effect on cerebral infarct size and no effect on blood pressure. BB3 increased HGF receptor c-Met phosphorylation and synaptophysin expression in penumbral tissue consistent with a neurorestorative mechanism from HGF-like activity. In mouse tMCAO, BB3 starting 10 minutes after reperfusion and continued for 14 days improved neurologic function that persisted for 8 weeks in some, but not all measures. Study in animals with comorbidities and those exposed to common stroke drugs are the next steps to complete preclinical assessment. These data, generated in independent, masked, and rigorously controlled settings, are the first to suggest that the HGF pathway can potentially be harnessed by BB3 for neurologic benefit after ischemic stroke.

Expression of neuronal protein synuclein gamma gene as a novel marker for breast cancer prognosis.

Synucleins are emerging as central players in the fundamental neural processes and in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases. However, synuclein gamma (SNCG), previously identified as a breast cancer specific gene (BCSG1), is also highly expressed in breast carcinomas, but not expressed in normal or benign breast tissues. We analyzed SNCG gene expression in 93 clinical breast specimens and associated it with clinical outcome. Overall SNCG mRNA expression was detectable in 36% breast cancers. However, 81% of stage III/IV breast cancers were positive for SNCG expression, while only 15% of stage I/II breast cancers were positive for SNCG expression. In contrast, SNCG was undetectable in benign breast lesions. Expression of SNCG in the primary tumor also significantly associated with lymph node involvement and metastasis. There was no significant correlation between SNCG gene expression and age, menstruation, and status of ER, PR, PCNA, and HER-2. Patients whose tumors expressed SNCG had a significantly shorter DFS and a high probability of death when compared with those whose tumors did not express SNCG. The hazard ratio of metastasis or recurrence according to the SNCG status was 4.515 (95% CI, 1,188-17.154; P = 0.027). Cox multivariate analysis showed that SNCG had independent prognostic significance above and beyond conventional variables. This study suggests that the expression of SNCG is an independent predictive marker for recurrence and metastasis in breast cancer progression. SNCG is expected to be a useful marker for breast cancer progression and a potential target for breast cancer treatment.

Identification of genes that modulate sensitivity of U373MG glioblastoma cells to cis-platinum.

Scatter factor (hepatocyte growth factor) and its receptor c-Met are increasingly expressed during progression from low-grade to high-grade gliomas. Scatter factor/c-Met signaling induces glioma cell motility, invasion, angiogenesis and resistance to DNA-damaging agents. The latter is relevant to the understanding of the resistance of human gliomas to chemotherapy and radiotherapy. The goal of this study was to identify a set of genes that may contribute to scatter factor-mediated protection of U373MG cells against cis-platinum, a DNA cross-linking agent. We used DNA microarray assays, confirmatory semiquantitative reverse transcription-polymerase chain reaction analysis and functional assays to identify genes involved in the scatter factor-induced resistance of U373MG to cis-platinum. We identified a group of genes that are overexpressed in cells treated with scatter factor plus cis-platinum relative to cells treated with cis-platinum alone and confirmed some of these gene expression alterations by reverse transcription-polymerase chain reaction. Inhibiting the expression of three of these genes--polycystic kidney disease 1, amplified in breast cancer 1 and DEAD/H box helicase 21--using small interfering RNAs reduced survival of cis-platinum-treated cells and partially reversed the scatter factor protection against cis-platinum. Dominant-negative Akt and IkappaB super-repressor expression vectors inhibited the scatter factor protection, and abrogated the ability of scatter factor to alter the expression of DEAD/H box helicase 21 and polycystin (PKD1) within the context of cis-platinum exposure. The Akt and nuclear factor-kappaB inhibitors had no effect on amplified in breast cancer 1 expression. These studies implicate DEAD/H box helicase 21, polycystin (PKD1) and amplified in breast cancer 1 as novel transcription-dependent regulators of scatter factor-mediated glioma cell protection against cytotoxic death, and identify other potential regulators for future study.

Role of NF-kappaB signaling in hepatocyte growth factor/scatter factor-mediated cell protection.

The cytokine scatter factor/hepatocyte growth factor (HGF/SF) protects epithelial, carcinoma, and other cell types against cytotoxicity and apoptosis induced by DNA-damaging agents such as ionizing radiation and adriamycin (ADR, a topoisomerase IIalpha inhibitor). We investigated the role of nuclear factor kappa B (NF-kappaB) signaling in HGF/SF-mediated protection of human prostate cancer (DU-145) and Madin-Darby canine kidney (MDCK) epithelial cells against ADR. HGF/SF caused the rapid nuclear translocation of the p65 (RelA) subunit of NF-kappaB associated with the transient loss of the inhibitory subunit IkappaB-alpha. Exposure to HGF/SF caused the activation of an NF-kappaB luciferase reporter that was blocked or attenuated by the expression of a mutant 'super-repressor' IkappaB-alpha. Electrophoretic mobility shift assay supershift assays revealed that HGF/SF treatment induced the transient binding of various NF-kappaB family proteins (p65, p50, c-Rel, and RelB) with radiolabeled NF-kappaB-binding oligonucleotides. The HGF/SF-mediated protection of DU-145 and MDCK cells against ADR (demonstrated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays) was abrogated by the IkappaB-alpha super-repressor. The ability of HGF/SF to activate NF-kappaB signaling was dependent on c-Akt --> Pak1 (p21-associated kinase-1) signaling (with Pak1 downstream of c-Akt) and was inhibited by the tumor suppressor PTEN (phosphatase and tensin homolog). Inhibitors of phosphatidylinositol-3'-kinase and Src family kinases significantly inhibited HGF/SF-mediated activation of NF-kappaB, while inhibitors of MEK, protein kinase C, and p70 S6 kinase had a modest effect or no effect on NF-kappaB activity. HGF/SF induced the expression of several known NF-kappaB target genes (cIAP-1 (cellular inhibitor of apoptosis-1), cIAP-2, and TRAF-2 (TNF receptor-associated factor-2)) in an NF-kappaB-dependent manner; HGF/SF blocked the inhibition of expression of these genes by ADR. Experimental manipulation of expression of these genes suggests that they (particularly TRAF-2 and cIAP-2) contribute to the protection against ADR by HGF/SF. These findings suggest that HGF/SF activates NF-kappaB through a c-Akt --> Pak1 signaling pathway that is also dependent on Src, and that NF-kappaB contributes to HGF/SF-mediated protection against ADR.

Structural determinants of the BRCA1 : estrogen receptor interaction.

Previously, we showed that the BRCA1 protein interacts directly and functionally with estrogen receptor-alpha (ER-alpha), resulting in the inhibition of estradiol (E2)-stimulated ER-alpha transcriptional activity. The interaction sites were mapped to the N-terminus of BRCA1 (within amino acids (aa) 1-302) and the ligand-binding domain/activation function-2 (LBD/AF-2) region (within aa 282-420) of ER-alpha. In this study, we have further characterized the structure/function relationship for the BRCA1 : ER-alpha interaction. We found that the N-terminal RING domain (aa 20-64) is not required for the BRCA1 : ER-alpha interaction. We identified two separate contact points for ER-alpha, one within aa 1-100 and the other within aa 100-200 of BRCA1; and we showed that each of these BRCA1 peptides interacts with BRCA1 in vitro and in vivo. By using different fragments of the BRCA1 N-terminus, we found that aa 67-100 and 101-133 are required for the interaction with ER-alpha, but that aa 1-67 and 134-302 are dispensible. Previously, we showed that BRCA1 aa 1-302 does not inhibit E2-stimulated ER-alpha transcriptional activity but does bind to ER-alpha and acts as a dominant negative inhibitor of the full-length BRCA1 protein. Somewhat surprisingly, we found that BRCA1 aa 1-100 and BRCA1 aa 101-200 (but not aa 201-300) each inhibited ER-alpha activity, although not as efficiently as full-length BRCA1. Mutations within an HIV Rev-like nuclear export signal that resembles a nuclear receptor corepressor motif (aa 86-95) impaired the ability of both truncated (aa 1-100) and full-length (aa 1-1863) BRCA1 proteins to interact with and/or repress ER-alpha activity. Based on these findings, a partial BRCA1 : ER-alpha three-dimensional structure is proposed. The implications of these findings for understanding the BRCA1 : ER-alpha interaction are discussed.