IL-6 Trans-Signaling Is Increased in Diabetes, Impacted by Glucolipotoxicity, and Associated With Liver Stiffness and Fibrosis in Fatty Liver Disease.

Many people living with diabetes also have nonalcoholic fatty liver disease (NAFLD). Interleukin-6 (IL-6) is involved in both diseases, interacting with both membrane-bound (classical) and circulating (trans-signaling) soluble receptors. We investigated whether secretion of IL-6 trans-signaling coreceptors are altered in NAFLD by diabetes and whether this might associate with the severity of fatty liver disease. Secretion patterns were investigated with use of human hepatocyte, stellate, and monocyte cell lines. Associations with liver pathology were investigated in two patient cohorts: 1) biopsy-confirmed steatohepatitis and 2) class 3 obesity. We found that exposure of stellate cells to high glucose and palmitate increased IL-6 and soluble gp130 (sgp130) secretion. In line with this, plasma sgp130 in both patient cohorts positively correlated with HbA1c, and subjects with diabetes had higher circulating levels of IL-6 and trans-signaling coreceptors. Plasma sgp130 strongly correlated with liver stiffness and was significantly increased in subjects with F4 fibrosis stage. Monocyte activation was associated with reduced sIL-6R secretion. These data suggest that hyperglycemia and hyperlipidemia can directly impact IL-6 trans-signaling and that this may be linked to enhanced severity of NAFLD in patients with concomitant diabetes. ARTICLE HIGHLIGHTS: IL-6 and its circulating coreceptor sgp130 are increased in people with fatty liver disease and steatohepatitis. High glucose and lipids stimulated IL-6 and sgp130 secretion from hepatic stellate cells. sgp130 levels correlated with HbA1c, and diabetes concurrent with steatohepatitis further increased circulating levels of all IL-6 trans-signaling mediators. Circulating sgp130 positively correlated with liver stiffness and hepatic fibrosis. Metabolic stress to liver associated with fatty liver disease might shift the balance of IL-6 classical versus trans-signaling, promoting liver fibrosis that is accelerated by diabetes.

CdGAP maintains podocyte function and modulates focal adhesions in a Src kinase-dependent manner.

Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and ß-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with ß-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired ß-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury.

The Tetracycline-Controlled Transactivator (Tet-On/Off) System in ß-Cells Reduces Insulin Expression and Secretion in Mice.

Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in ß-cells, transgene silencing, low inducibility, ectopic expression, and off-targets effects are persistent challenges. In this study, we investigated whether an inducible Tetracycline (Tet)-Off system with ß-cell-specific mouse insulin promoter (MIP)-itTA-driven expression of tetracycline operon (TetO)-CreJaw/J could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination, ß-cell architecture, in vitro and in vivo glucose-stimulated insulin secretion, and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g., improved itTA, reverse rtTA, or tTA) in ß-cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with ß-cells and highlights the need for consistent and comprehensive controls when using the tetracycline-controlled transactivator systems (Tet-On or Tet-Off) for genome editing.

PGC-1α isoforms coordinate to balance hepatic metabolism and apoptosis in inflammatory environments.

OBJECTIVE: The liver is regularly exposed to changing metabolic and inflammatory environments. It must sense and adapt to metabolic need while balancing resources required to protect itself from insult. Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) is a transcriptional coactivator expressed as multiple, alternatively spliced variants transcribed from different promoters that coordinate metabolic adaptation and protect against inflammation. It is not known how PGC-1α integrates extracellular signals to balance metabolic and anti-inflammatory outcomes. METHODS: Primary mouse hepatocytes were used to evaluate the role(s) of different PGC-1α proteins in regulating hepatic metabolism and inflammatory signaling downstream of tumor necrosis factor alpha (TNFα). Gene expression and signaling analysis were combined with biochemical measurement of apoptosis using gain- and loss-of-function in vitro and in vivo. RESULTS: Hepatocytes expressed multiple isoforms of PGC-1α, including PGC-1α4, which microarray analysis showed had common and isoform-specific functions linked to metabolism and inflammation compared with canonical PGC-1α1. Whereas PGC-1α1 primarily impacted gene programs of nutrient metabolism and mitochondrial biology, TNFα signaling showed several pathways related to innate immunity and cell death downstream of PGC-1α4. Gain- and loss-of-function models illustrated that PGC-1α4 uniquely enhanced expression of anti-apoptotic gene programs and attenuated hepatocyte apoptosis in response to TNFα or lipopolysaccharide (LPS). This was in contrast to PGC-1α1, which decreased the expression of a wide inflammatory gene network but did not prevent hepatocyte death in response to cytokines. CONCLUSIONS: PGC-1α variants have distinct, yet complementary roles in hepatic responses to metabolism and inflammation, and we identify PGC-1α4 as an important mitigator of apoptosis.

ARHGEF7 (ß-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function.

BACKGROUND: Previous studies showed that Cdc42, a member of the prototypical Rho family of small GTPases and a regulator of the actin cytoskeleton, is critical for the normal development and health of podocytes. However, upstream regulatory mechanisms for Cdc42 activity in podocytes are largely unknown. METHODS: We used a proximity-based ligation assay, BioID, to identify guanine nucleotide exchange factors that activate Cdc42 in immortalized human podocytes. We generated podocyte-specific ARHGEF7 (commonly known as ß-PIX) knockout mice by crossing ß-PIX floxed mice with Podocin-Cre mice. Using shRNA, we established cultured mouse podocytes with ß-PIX knockdown and their controls. RESULTS: We identified ß-PIX as a predominant guanine nucleotide exchange factor that interacts with Cdc42 in human podocytes. Podocyte-specific ß-PIX knockout mice developed progressive proteinuria and kidney failure with global or segmental glomerulosclerosis in adulthood. Glomerular podocyte density gradually decreased in podocyte-specific ß-PIX knockout mice, indicating podocyte loss. Compared with controls, glomeruli from podocyte-specific ß-PIX knockout mice and cultured mouse podocytes with ß-PIX knockdown exhibited significant reduction in Cdc42 activity. Loss of ß-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the prosurvival transcriptional regulator Yes-associated protein. CONCLUSIONS: These findings indicate that ß-PIX is required for the maintenance of podocyte architecture and glomerular function via Cdc42 and its downstream Yes-associated protein activities. This appears to be the first evidence that a Rho-guanine nucleotide exchange factor plays a critical role in podocytes.

Recessive mutation in CD2AP causes focal segmental glomerulosclerosis in humans and mice.

Although sequence variants in CD2-associated protein (CD2AP) have been identified in patients with focal segmental glomerulosclerosis (FSGS), definitive proof of causality in human disease is meager. By whole-exome sequencing, we identified a homozygous frame-shift mutation in CD2AP (p.S198fs) in three siblings born of consanguineous parents who developed childhood-onset FSGS and end stage renal disease. When the same frameshift mutation was introduced in mice by gene editing, the mice developed FSGS and kidney failure. These results provide conclusive evidence that homozygous mutation of CD2AP causes FSGS in humans.

Peptide-based sequestration of the adaptor protein Nck1 in pancreatic ß cells enhances insulin biogenesis and protects against diabetogenic stresses.

One feature of diabetes is the failure of pancreatic ß cells to produce insulin, but the molecular mechanisms leading to this failure remain unclear. Increasing evidence supports a role for protein kinase R-like endoplasmic reticulum kinase (PERK) in the development and function of healthy pancreatic ß cells. Previously, our group identified the adaptor protein Nck1 as a negative regulator of PERK. Indeed, we demonstrated that Nck1, by directly binding PERK autophosphorylated on Tyr561, limits PERK activation and signaling. Accordingly, we found that stable depletion of Nck1 in ß cells promotes PERK activation and signaling, increases insulin biosynthesis, and improves cell viability in response to diabetes-related stresses. Herein, we explored the therapeutic potential of abrogating the interaction between Nck and PERK to improve ß-cell function and survival. To do so, we designed and used a peptide containing the minimal PERK sequence involved in binding Nck1 conjugated to the cell-permeable protein transduction domain from the HIV protein TAT. In the current study, we confirm that the synthetic TAT-Tyr(P)561 phosphopeptide specifically binds the SH2 domain of Nck and prevents Nck interaction with PERK, thereby promoting basal PERK activation. Moreover, we report that treatment of ß cells with TAT-Tyr(P)561 inhibits glucolipotoxicity-induced apoptosis, whereas it enhances insulin production and secretion. Taken together, our results support the potential of sequestering Nck using a synthetic peptide to enhance basal PERK activation and create more robust ß cells.

The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes.

Nephrotic syndrome is a kidney disease featured by heavy proteinuria. It is caused by injury to the specialized epithelial cells called "podocytes" within the filtration unit of the kidney, glomerulus. Previous studies showed that hyperactivation of the RhoGTPase, Rac1, in podocytes causes podocyte injury and glomerulosclerosis (accumulation of extracellular matrix in the glomerulus). However, the mechanism by which Rac1 is activated during podocyte injury is unknown. Trio is a guanine nucleotide exchange factor (GEF) known to activate Rac1. By RNA-sequencing, we found that Trio mRNA is abundantly expressed in cultured human podocytes. Trio mRNA was also significantly upregulated in humans with minimal change disease and focal segmental glomerulosclerosis, two representative causes of nephrotic syndrome. Reduced expression of Trio in cultured human podocytes decreased basal Rac1 activity, cell size, attachment to laminin, and motility. Furthermore, while the pro-fibrotic cytokine, transforming growth factor ß1 increased Rac1 activity in control cells, it decreases Rac1 activity in cells with reduced Trio expression. This was likely due to simultaneous activation of the Rac1-GTPase activation protein, CdGAP. Thus, Trio is important in the basal functions of podocytes and may also contribute to glomerular pathology, such as sclerosis, via Rac1 activation.

Rac1 activation in podocytes induces the spectrum of nephrotic syndrome.

Hyper-activation of Rac1, a small GTPase, in glomerular podocytes has been implicated in the pathogenesis of familial proteinuric kidney diseases. However, the role of Rac1 in acquired nephrotic syndrome is unknown. To gain direct insights into this, we generated a transgenic mouse model expressing a doxycycline-inducible constitutively active form of Rac1 (CA-Rac1) in podocytes. Regardless of the copy number, proteinuria occurred rapidly within five days, and the histology resembled minimal change disease. The degree and severity of proteinuria were dependent on the transgene copy number. Upon doxycycline withdrawal, proteinuria resolved completely (one copy) or nearly completely (two copy). After one month of doxycycline treatment, two-copy mice developed glomerulosclerosis that resembled focal segmental glomerulosclerosis (FSGS) with urinary shedding of transgene-expressing podocytes. p38 MAPK was activated in podocytes upon CA-Rac1 induction while a p38 inhibitor attenuated proteinuria, podocyte loss, and glomerulosclerosis. Mechanistically, activation of Rac1 in cultured mouse podocytes reduced adhesiveness to laminin and induced redistribution of ß1 integrin, and both were partially reversed by the p38 inhibitor. Activation of Rac1 in podocytes was also seen in kidney biopsies from patients with minimal change disease and idiopathic FSGS by immunofluorescence while sera from the same patients activated Rac1 in cultured human podocytes. Thus, activation of Rac1 in podocytes causes a spectrum of disease ranging from minimal change disease to FSGS, due to podocyte detachment from the glomerular basement membrane that is partially dependent on p38 MAPK.

Binding and inhibition of the ternary complex factor Elk-4/Sap1 by the adapter protein Dok-4.

The adapter protein Dok-4 (downstream of kinase-4) has been reported as both an activator and inhibitor of Erk and Elk-1, but lack of knowledge about the identity of its partner molecules has precluded any mechanistic insight into these seemingly conflicting properties. We report that Dok-4 interacts with the transactivation domain of Elk-4 through an atypical phosphotyrosine-binding domain-mediated interaction. Dok-4 possesses a nuclear export signal and can relocalize Elk-4 from nucleus to cytosol, whereas Elk-4 possesses two nuclear localization signals that restrict interaction with Dok-4. The Elk-4 protein, unlike Elk-1, is highly unstable in the presence of Dok-4, through both an interaction-dependent mechanism and a pleckstrin homology domain-dependent but interaction-independent mechanism. This is reversed by proteasome inhibition, depletion of endogenous Dok-4 or lysine-to-arginine mutation of putative Elk-4 ubiquitination sites. Finally, Elk-4 transactivation is potently inhibited by Dok-4 overexpression but enhanced by Dok-4 knockdown in MDCK renal tubular cells, which correlates with increased basal and EGF-induced expression of Egr-1, Fos and cylcinD1 mRNA, and cell proliferation despite reduced Erk activation. Thus, Dok-4 can target Elk-4 activity through multiple mechanisms, including binding of the transactivation domain, nuclear exclusion and protein destabilization, without a requirement for inhibition of Erk.

Disease-causing mutations of RhoGDIα induce Rac1 hyperactivation in podocytes.

Nephrotic syndrome (NS) describes a group of kidney disorders in which there is injury to podocyte cells, specialized cells within the kidney's glomerular filtration barrier, allowing proteins to leak into the urine. Three mutations in ARHGDIA, which encodes Rho GDP dissociation inhibitor α (GDIα), have been reported in patients with heritable NS and encode the following amino acid changes: ΔD185, R120X, and G173V. To investigate the impact of these mutations on podocyte function, endogenous GDIα was knocked-down in cultured podocytes by shRNA and then the cells were re-transfected with wild-type or mutant GDIα constructs. Among the 3 prototypical Rho-GTPases, Rac1 was markedly hyperactivated in podocytes with any of the 3 mutant forms of GDIα while the activation of RhoA and Cdc42 was modest and variable. All three mutant GDIα proteins resulted in slow podocyte motility, suggesting that podocytes are sensitive to the relative balance of Rho-GTPase activity. In ΔD185 podocytes, both random and directional movements were impaired and kymograph analysis of the leading edge showed increased protrusion and retraction of leading edge (phase switching). The mutant podocytes also showed impaired actin polymerization, smaller cell size, and increased cellular projections. In the developing kidney, GDIα expression increased as podocytes matured. Conversely, active Rac1 was detected only in immature, but not in mature, podocytes. The results indicate that GDIα has a critical role in suppressing Rac1 activity in mature podocytes, to prevent podocyte injury and nephrotic syndrome.

Loss of Rho-GDIα sensitizes podocytes to lipopolysaccharide-mediated injury.

Nephrotic syndrome is a disease of glomerular permselectivity that can arise as a consequence of heritable or acquired changes to the integrity of the glomerular filtration barrier. We recently reported two siblings with heritable nephrotic syndrome caused by a loss of function mutation in the gene ARHGDIA, which encodes for Rho guanine nucleotide dissociation inhibitor-α (GDIα). GDIs are known to negatively regulate Rho-GTPase signaling. We hypothesized that loss of GDIα sensitizes podocytes to external injury via hyperactivation of Rho-GTPases and p38 MAPK. We examined the response of cultured podocytes with and without knockdown of GDIα to LPS injury by assessing the levels of phospho-p38 as well as the degree of synaptopodin loss. GDIα knockdown podocytes showed more pronounced and sustained p38 phosphorylation in response to LPS compared with control podocytes, and this was blunted significantly by the Rac1 inhibitor. In LPS-treated control podocytes, synaptopodin degradation occurred, and this was dependent on p38, the proteasome, and cathepsin L. In GDIα knockdown podocytes, the same events were triggered, but the levels of synaptopodin after LPS treatment were significantly lower than in control podocytes. These experiments reveal a common pathway by which heritable and environmental risk factors converge to injure podocytes, from Rac1 hyperactivation to p38 phosphorylation and synaptopodin degradation via the ubiquitin-proteasome pathway and cathepsin L.

Protein tyrosine phosphatase 1B inhibition protects against podocyte injury and proteinuria.

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed nonreceptor protein-tyrosine phosphatase that regulates various cellular functions, including migration. Recent studies suggest that an increased migratory phenotype of podocytes may be responsible for proteinuria and foot process effacement. The current study addresses the role of PTP1B in podocyte injury and proteinuria. PTP1B was markedly up-regulated in the glomerulus, notably in podocytes, in three rodent models of podocyte injury. Podocyte-specific ablation of the PTP1B gene ameliorated proteinuria induced by lipopolysaccharide and Adriamycin (doxorubicin). The use of a specific PTP1B inhibitor also protected against lipopolysaccharide-induced proteinuria. In contrast, podocyte-specific PTP1B transgenic male mice developed spontaneous proteinuria and foot process effacement. In cultured mouse podocytes, PTP1B knockdown and/or pretreatment with the PTP1B inhibitor blunted lipopolysaccharide-induced cell migration, activation of Src-family kinases (SFKs), and phosphorylation of focal adhesion kinase at Y397 (pFAK(Y397)), the latter being crucial for cell migration. Lipopolysaccharide-injected mice showed increased glomerular expression of active SFKs and pFAK(Y397), both of which were inhibited by podocyte-specific PTP1B knockout and the PTP1B inhibitor. Moreover, podocyte-specific PTP1B transgenic mice showed increased glomerular expression of active SFKs and pFAK(Y397). In summary, PTP1B up-regulation in podocytes induces a migratory response by activating SFKs and FAK, leading to foot process effacement and proteinuria. Pharmacological inhibition of PTP1B may have therapeutic potential in the treatment of proteinuric diseases.

Planar cell polarity pathway regulates nephrin endocytosis in developing podocytes.

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/ß-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.

Nuclear factor of activated T cells mediates RhoA-induced fibronectin upregulation in glomerular podocytes.

Glomerulosclerosis is featured by accumulation of the extracellular matrixes in the glomerulus. We showed previously that activation of the small GTPase RhoA in podocytes induces heavy proteinuria and glomerulosclerosis in the mouse. In the current study, we investigated the mechanism by which RhoA stimulates the production of one of the extracellular matrixes, fibronectin, by podocytes, specifically testing the role of nuclear factor of activated T cells (NFAT). Expression of constitutively active RhoA in cultured podocytes activated the fibronectin promoter, upregulated fibronectin protein, and activated NFAT. Expression of constitutively active NFAT in podocytes also activated the fibronectin promoter and upregulated fibronectin protein. RhoA-induced NFAT activation and fibronectin upregulation were both dependent on the calcium/calmodulin pathway and Rho kinase. NFAT activation was also observed in vivo in the rat and mouse models of podocyte injury and proteinuria, and NFAT inhibition ameliorated fibronectin upregulation in the latter. RhoA activation induced a rise of intracellular calcium ion concentration ([Ca(2+)]i), which was at least in part dependent on the transient receptor potential canonical 6 (TRPC6) cation channel. The results indicate that RhoA activates NFAT by inducing a rise of [Ca(2+)]i in podocytes, which in turn contributes to fibronectin upregulation. This pathway may be responsible for the pathogenesis of certain glomerular diseases such as hypertension-mediated glomerulosclerosis.

ARHGDIA: a novel gene implicated in nephrotic syndrome.

BACKGROUND: Congenital nephrotic syndrome arises from a defect in the glomerular filtration barrier that permits the unrestricted passage of protein across the barrier, resulting in proteinuria, hypoalbuminaemia, and severe oedema. While most cases are due to mutations in one of five genes, in up to 15% of cases, a genetic cause is not identified. We investigated two sisters with a presumed recessive form of congenital nephrotic syndrome. METHODS AND RESULTS: Whole exome sequencing identified five genes with diallelic mutations that were shared by the sisters, and Sanger sequencing revealed that ARHGDIA that encodes Rho GDP (guanosine diphosphate) dissociation inhibitor α (RhoGDIα, OMIM 601925) was the most likely candidate. Mice with targeted inactivation of ARHGDIA are known to develop severe proteinuria and nephrotic syndrome, therefore this gene was pursued in functional studies. The sisters harbour a homozygous in-frame deletion that is predicted to remove a highly conserved aspartic acid residue within the interface where the protein, RhoGDIα, interacts with the Rho family of small GTPases (c.553_555del(p.Asp185del)). Rho-GTPases are critical regulators of the actin cytoskeleton and when bound to RhoGDIα, they are sequestered in an inactive, cytosolic pool. In the mouse kidney, RhoGDIα was highly expressed in podocytes, a critical cell within the glomerular filtration barrier. When transfected in HEK293T cells, the mutant RhoGDIα was unable to bind to the Rho-GTPases, RhoA, Rac1, and Cdc42, unlike the wild-type construct. When RhoGDIα was knocked down in podocytes, RhoA, Rac1, and Cdc42 were hyperactivated and podocyte motility was impaired. The proband's fibroblasts demonstrated mislocalisation of RhoGDIα to the nucleus, hyperactivation of the three Rho-GTPases, and impaired cell motility, suggesting that the in-frame deletion leads to a loss of function. CONCLUSIONS: Mutations in ARHGDIA need to be considered in the aetiology of heritable forms of nephrotic syndrome.

New insights into Dok-4 PTB domain structure and function.

The seven members of the Dok adapter protein family share a highly conserved phosphotyrosine-binding (PTB) domain. In the case of Dok-1, 2 and 3, the PTB domain binds to the lipid phosphatase Ship1, a key component of their inhibitory signaling mechanisms in immune cells. In contrast to most other Dok family members, Dok-4 is expressed widely but is poorly understood, largely because of limited knowledge of its partner molecules. We previously showed that, in contrast to the Dok-1 PTB domain (defined as aa 107-260), the homologous sequence in Dok-4 (aa 100-233) bound very poorly to Ret, a known Dok-4 partner. In the current study, we show that binding of Dok-4 to Ret requires residues C-terminal to the previously defined PTB domain boundaries (up to aa 246). These residues are predicted to form an extension in a critical C-terminal α-helix. We show that the Dok-4 PTB domain also binds the phosphorylated NPXY motifs in Ship1 but not Ship2. Finally, we found that a rare human single nucleotide polymorphism causing a R186H substitution in the PTB domain abolishes tyrosine phosphorylation of Dok-4 by Ret. In addition to providing a clearer understanding of Dok-4 PTB domain structure and function, our findings point to a potential mechanism for Dok-4 inhibitory signaling in T-cells and to the possibility of a rare Dok-4-related phenotype in humans.

Basal and Src kinase-mediated activation of the EphA2 promoter requires a cAMP-responsive element but is CREB-independent.

We have previously identified the EphA2 receptor tyrosine kinase as a potentially important injury-responsive gene and a transcriptional target of Src kinase activity in renal ischemia-reperfusion injury (IRI). In the present study, we confirmed, using EphA2 gene trap mice that the endogenous EphA2 promoter is strongly activated following renal IRI. We also examined in more detail the mechanisms responsible for Src kinase-induced activation of the -2 kb human EphA2 promoter and found that the minimal Src-responsive elements were contained in the -145 to +137 region of the human EphA2 gene. This region contains a canonical cAMP-responsive element (CRE) that we found to be critical for both basal and Src kinase-induced transcriptional activity. However, despite activation of the prototypical CRE-binding factor CREB by the Src kinase Fyn, siRNA-mediated knockdown of CREB had no significant impact on either basal or Fyn-induced EphA2 promoter activity. Similarly, activation of CREB by the adenylate cyclase agonist forskolin failed to induce EphA2 promoter activation. Thus, Src kinase-induced activation of the EphA2 promoter is CRE-dependent but CREB-independent.

Probiotic Lactobacillus acidophilus and L. casei mix sensitize colorectal tumoral cells to 5-fluorouracil-induced apoptosis.

To assess the potential of Lactobacillus acidophilus and Lactobacillus casei strains to increase the apoptosis of a colorectal cancer cell line in the presence of 5-fluorouracil (5-FU), LS513 colorectal cancer cells were treated for 48 h with increasing concentrations of these lactic acid bacteria (LAB) in the presence of 100 mu g/ml of 5-FU. In the presence of 10(8) CFU/ml of live LAB, the apoptotic efficacy of the 5-FU increased by 40%, and the phenomenon was dose dependent. Moreover, irradiation-inactivated LAB caused the same level of induction, whereas microwave-inactivated LAB reduced the apoptotic capacity of the 5-FU. When cells were treated with a combination of live LAB and 5-FU, a faster activation of caspase-3 protein was observed, and the p21 protein seems to be downregulated. These results suggest that live L. acidophilus and L. casei are able to increase the apoptosis-induction capacity of 5-FU. The mechanisms of action are still not elucidated, and more research is needed to understand them. This is the first set of experiments demonstrating that some strains of LAB can enhance the apoptosis-induction capacity of the 5-FU. Based on these results, it is possible to speculate that LAB or probiotics could be used as an adjuvant treatment during anticancer chemotherapy.

Identification of Dok-4b, a Dok-4 splice variant with enhanced inhibitory properties.

Dok adapter proteins have been primarily implicated in negative regulation of tyrosine kinase signaling, but Dok-4 has been reported to exert both inhibitory and stimulatory effects. We have identified a splice variant of Dok-4, Dok-4b, which contains a 39 aa insert within the its C-terminal region. The approximately 45kDa Dok-4b protein was detected in several human epithelial cell lines. Based on genomic sequences, Dok-4b was also predicted to exist in primates and possibly bovines, but not in rodents or other species. Compared to Dok-4, Dok-4b inhibited the tyrosine kinase-induced activation of both Erk and Elk-1 more strongly. Truncation of the C-terminal region of Dok-4 (Dok-4 DeltaCT) also enhanced the inhibitory activity of Dok-4, whereas expression of the isolated C-terminal domain enhanced Elk-1 activation, suggesting that the N-terminus and C-terminus of Dok-4 possess opposing inhibitory and stimulatory properties, respectively, the balance of which is altered by alternative splicing of Dok-4 to Dok-b.