Thioredoxin-interacting protein is a biomechanical regulator of Src activity: key role in endothelial cell stress fiber formation.

RATIONALE: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell-cell junction integrity. A key mediator of steady flow-induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. OBJECTIVE: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its α-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. METHODS AND RESULTS: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2-vascular endothelial cadherin interaction. CONCLUSIONS: In summary, these data characterize a fluid shear stress-mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin-Src homology phosphatase-2-Src pathway.

Thioredoxin-interacting protein mediates nuclear-to-plasma membrane communication: role in vascular endothelial growth factor 2 signaling.

OBJECTIVE: Thioredoxin-interacting protein (TXNIP) and poly-ADP-ribose polymerase 1 (PARP1) are both regulated by changes in cellular reduction-oxidation (redox) state and localize to the nucleus basally in human umbilical vein endothelial cells (HUVEC). Previously we showed a novel mechanism for PARP1 inhibition-mediated HUVEC survival through activation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling in response to stress-induced apoptosis. In addition, we showed TXNIP translocation to the plasma membrane (PM) and activation of VEGFR2 in response to physiological stimuli. Because TXNIP is an α-arrestin that regulates VEGFR2 signaling, we hypothesized that PARP1 regulates TXNIP localization and function that might affect HUVEC stress-induced apoptosis. METHODS AND RESULTS: HUVEC treated with 10 µmol/L PARP1 inhibitor (PJ34) were protected from TNF (10 ng/mL) or H(2)O(2) (300 µmol/L) mediated cell death. HUVEC transfected with TXNIP siRNA lost the protective effect of PARP1 inhibition, suggesting a protective role for TXNIP. Using immunofluorescence, cell fractionation analysis, and plasma membrane sheet assay, TXNIP was shown to translocate to the plasma membrane after PARP1 inhibition. TXNIP translocation was associated with activation of VEGFR2 signaling. Functionally, TXNIP-PARP1 interaction was decreased on PJ34 treatment, suggesting PARP1 as a novel regulator of TXNIP localization and function. CONCLUSIONS: These findings demonstrate a novel regulatory mechanism of TXNIP by PARP1 to mediate activation of plasma membrane signaling and cell survival.

Thioredoxin-interacting protein mediates TRX1 translocation to the plasma membrane in response to tumor necrosis factor-α: a key mechanism for vascular endothelial growth factor receptor-2 transactivation by reactive oxygen species.

OBJECTIVE: Thioredoxin-interacting protein (TXNIP) promotes inflammation in endothelial cells (EC) by binding to thioredoxin-1 (TRX1) in a redox-dependent manner. Formation of the TXNIP-TRX1 complex relieves inhibition of the apoptosis signal-regulating kinase 1-c-Jun N-terminal kinase-vascular cell adhesion molecule-1 pathway. Because TXNIP is an α-arrestin with numerous protein-protein interacting domains, we hypothesized that TXNIP-TRX1 trafficking should alter function of EC exposed to reactive oxygen species (ROS). METHODS AND RESULTS: In response to physiological levels of ROS (10 ng/mL tumor necrosis factor-α and 30 µmol/L H(2)O(2)), TXNIP-TRX1 translocated to the plasma membrane in human umbilical vein EC, with a peak at 30 minutes, as measured by immunofluorescence colocalization with vascular endothelial-cadherin, cell fractionation, and membrane sheet assay. TXNIP-mediated translocation of TRX1 to the membrane required TXNIP and TRX1 binding, as evidenced by inability of the ROS-insensitive TXNIP-Cys247Ser mutant to promote membrane localization. Vascular endothelial growth factor signaling required TXNIP, as shown by significant decreases in plasma membrane tyrosine phosphorylation and EC migration after TRX1 knockdown. Furthermore, TXNIP knockdown increased human umbilical vein EC apoptosis induced by tumor necrosis factor. Rescue with TXNIP-wild-type but not TXNIP-Cys247Ser prevented cell death. CONCLUSIONS: These findings suggest a novel role for the TXNIP-TRX1 complex to enable inflammation by promoting EC survival and vascular endothelial growth factor signaling under conditions of physiological oxidative stress.

Redox redux: protecting the ischemic myocardium.

Cardiac ischemia-reperfusion (I-R) injury occurs upon prompt restoration of blood flow to the ischemic myocardium after an acute myocardial infarction. Interestingly, many of the features of I-R injury are related to impaired mitochondrial signaling and mitochondrial dysfunction. Restoring cardiac energy bioavailability and reduction-oxidation (redox) signaling are therefore important in recovery after I-R injury. In this issue of the JCI, Yoshioka and colleagues describe an important and unexpected role for thioredoxin-interacting protein (TXNIP) in the control of mitochondrial respiration and cell energy metabolism. Their findings could open the door for development of TXNIP-targeted therapeutic approaches for the treatment of cardiac I-R injury.

Thioredoxin interacting protein: redox dependent and independent regulatory mechanisms.

SIGNIFICANCE: The thioredoxin-interacting protein (TXNIP, also termed VDUP1 for vitamin D upregulated protein or TBP2 for thioredoxin-binding protein) was originally discovered by virtue of its strong regulation by vitamin D. Recently, TXNIP has been found to regulate the cellular reduction-oxidation (redox) state by binding to and inhibiting thioredoxin (TRX) in a redox-dependent fashion. RECENT ADVANCES: Studies of the Hcb-19 mouse, TXNIP nonsense mutated mouse, demonstrate redox-mediated roles in lipid and glucose metabolism, cardiac function, inflammation, and carcinogenesis. Exciting recent data indicate important roles for TXNIP in redox independent signaling. Specifically, sequence analysis revealed that TXNIP is a member of the classical visual/ß-arrestin superfamily, and is one of the six members of the arrestin domain-containing (ARRDC, or α-arrestin) family. CRITICAL ISSUES: Although the function of α-arrestins is not well known, recent studies suggest roles in endocytosis and protein ubiquitination through PPxY motifs in their C-terminal tails. Importantly, the ability of TXNIP to inhibit glucose uptake was found to be independent of TRX binding. Further investigation showed that several metabolic functions of TXNIP were due to the arrestin domains, thus further supporting the importance of redox independent functions of TXNIP. FUTURE DIRECTIONS: Since TXNIP transcription and protein stability are highly regulated by multiple tissue-specific stimuli, it appears that TXNIP should be a good therapeutic target for metabolic diseases.