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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:906.)
© 2008 American Heart Association, Inc.

Cell Biology and Signaling

Human Stanniocalcin-1 Blocks TNF-–Induced Monolayer Permeability in Human Coronary Artery Endothelial Cells

Changyi Chen; Md Saha Jamaluddin; Shaoyu Yan; David Sheikh-Hamad; Qizhi Yao

From the Molecular Surgeon Research Center (C.C., M.S.J., S.Y., Q.Y.), Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery; and the Renal Section (D.S.-H.), Department of Medicine, Baylor College of Medicine, Houston, Tex.

Correspondence to Changyi (Johnny) Chen, MD, PhD, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, One Baylor Plaza, Mail stop: NAB-2010, Houston, TX 77030. E-mail jchen{at}bcm.tmc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— Our previous studies revealed upregulation of stanniocalcin-1 (STC1) in cardiac vessels in dilated cardiomyopathy. However, the functional significance of STC1 is unknown. The objective of this study was to determine the effects of STC1 on TNF-–induced monolayer permeability of human coronary artery endothelial cells (HCAECs).

Methods and Results— Cells were pretreated with STC1 for 30 minutes followed by treatment with TNF- (2 ng/mL) for 24 hours. Monolayer permeability was studied using a transwell system. STC1 pretreatment significantly blocked TNF-–induced monolayer permeability in a concentration- and time-dependent manner. STC1 effectively blocked TNF-–induced downregulation of endothelial tight junction proteins zonula occluden-1 and claudin-1 at both mRNA and protein levels. STC1 also significantly decreased TNF-–induced superoxide anion production. The inhibitory effect of STC1 was specific to TNF-, as it failed to inhibit VEGF-induced endothelial permeability. Furthermore, STC1 partially blocked NF-B and JNK activation in TNF-–treated endothelial cells. JNK inhibitor and antioxidant also effectively blocked TNF-–induced NF-B activation and monolayer permeability in HCAECs.

Conclusions— STC1 maintains endothelial permeability in TNF-–treated HCAECs through preservation of tight junction protein expression, suppression of superoxide anion production, and inhibition of the activation of NFB and JNK, suggesting an important role for STC1 in regulating endothelial functions during cardiovascular inflammation.

Key Words: stanniocalcin-1 • TNF- • endothelial cell • permeability • superoxide anion

	Introduction

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Stanniocalcin 1 (STC1) is a 25-kDa disulfide-linked homodimeric glycoprotein, which was originally identified as a secretory hormone of the corpuscles of Stannius, an endocrine gland unique to bony fish.¹ The mammalian homolog of STC1 has been found in many species including humans, rats, and mice.^2,3 Human STC1 is encoded by a single copy gene located on chromosome 8p11.2-p21 and comprises 4 exons that encode 247 amino acids with 11 cysteine residues.^2,3 Human and mouse STC1 proteins have 98% amino acid identity and share 73% overall sequence homology to fish STC.² Unlike the localized expression of STC1 in fish, mammalian STC1 is not detected in the blood and is expressed in many organs including the kidney, intestine, heart, thyroid, lung, placenta, brain, and bone,^4,5 suggesting a paracrine rather than an endocrine role for STC1 in mammals. The primary function of STC in fish is to prevent hypercalcemia⁶ by inhibiting calcium influx through the gills and gut and stimulating phosphate reabsorption by the kidneys.⁷ Similarly, in rodents, STC1 reduces the intestinal uptake of calcium while enhancing that of phosphate.⁸ Furthermore, this gene seems to play diverse roles in numerous developmental, physiological, and pathological processes including cancer, pregnancy, lactation, angiogenesis, organogenesis, cerebral ischemia, and hypertonic stress.⁹ Studies also indicate that STC1 modulates inflammation. For example, STC1 inhibited chemotaxis of mouse macrophages and human monoblasts in response to monocyte chemotactic protein-1 (MCP-1) and stromal cell-derived factor-1 alpha (SDF1-)¹⁰ and decreased transmigration of macrophages and T-lymphocytes across quiescent or interleukin (IL)-1 beta (IL-1β)–activated human endothelial cells.¹¹ Despite the growing body of knowledge about STC1, little is known about its effects on the vascular system and how it might affect endothelial barrier function during inflammation.

The endothelial barrier is established and maintained mainly by endothelium-endothelium junction structures including adherens junctions, tight junctions (TJ), desmosomes, and gap junctions. Cohesive interactions between TJ molecules localized on apposing strands occlude the lateral intercellular space, thus restricting the paracellular diffusion of solutes. TJ are composed of integral membrane proteins including occludin and claudins; junctional adhesion molecule-A (JAM-A); and intracellular proteins such as zonula occluden-1 (ZO-1) and cingulin.¹² Increased vascular permeability is a common feature in many pathological conditions such as obstruction of airways during asthma and related pulmonary disorders,¹³ circulatory collapse in sepsis, cancer growth and metastatic spread,¹⁴ and many inflammatory conditions. Increased vascular permeability is also associated with the development of atherosclerosis.¹⁵ We have previously shown increased expression of STC1 in cardiac vessels and cardiomyocytes of patients with dilated cardiomyopathy.¹⁶ The expression of STC1 mRNA in endothelial cells and the remarkable increase in the expression of STC1 protein in blood vessels of the failing heart suggested an important role for this protein in the vascular system. Studies from our laboratories also suggest that STC1 suppresses inflammation^11,17; however, it is unknown whether STC1 plays a role in endothelial barrier function, particularly during inflammation. Because heart failure and atheresoclerosis are characterized by increased oxidative stress¹⁸ and upregulated expression of inflammatory cytokines such as TNF-, IL-1, and IL-6,¹⁹ we hypothesized that STC1 may inhibit cyctokine-induced endothelial permeability and thus may maintain endothelial integrity in the failing heart and atherosclerotic vessels. In this study, we examined the effects of STC1 on TNF-–induced permeability of human coronary artery endothelial cell (HCAEC) monolayer and determined the effects of STC1 on cytokine-induced changes in the expression of TJ molecules; generation of reactive oxygen species (ROS); and activation of mitogen-activated protein kinases (MAPKs) and transcription factor NF-kB. This study demonstrates a new role for STC1 in the regulation of inflammation and endothelial functions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemicals and Reagents
Recombinant human STC1 protein and rabbit antihuman STC1 antibodies were kindly provided by Dr Henrik Olsen, Human Genome Sciences (Rockville, Md).²⁰ STC1 protein was expressed in a baculovirus expression system and is greater than 90% pure.²¹ Endotoxin levels of the STC1 preparation are not detectable.²¹ Detail information about materials and methods is provided in the supplemental materials (available online at http://atvb.ahajournals.org).

Endothelial Permeability
Permeability indicated by paracellular influx across the HCAEC monolayer was studied in a Coaster Transwell system. They were pretreated with different concentrations of STC1 (6.25, 12.5, 25, 50, and 100 ng/mL) for 30 minutes and then treated with TNF- (2 ng/mL) or vehicle for up to 48 hours. In separate experiments, cells were treated with anti-STC1 antibody, VEGF, nifedipine, JNK inhibitor (SP600125), or SOD mimetic Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP).²² After treatment, an equal amount of Texas-Red-labeled dextran tracer was added to the upper chamber. The amount of tracer penetrating through the cell monolayer into the lower chamber was measured by a fluorometer.

Real-Time RT-PCR Analysis
The mRNA levels of ZO-1 and claudin-1 were determined by real-time PCR as described in our previous publication.²³ Expression for each target gene in each sample was normalized to β-actin.

Western Blot
Total cell extract, nuclear extract, or cytoplasmic extract of HCACEs was prepared. Equal amounts of proteins (50 µg) were loaded onto SDS-PAGE gel for electrophoresis, and proteins were transblotted overnight at 4°C onto Hybond-P polyvinylidene fluoride (PVDF) membrane and probed with the respective primary antibodies against human ZO-1, claudin-1, p-IB, total IB, NF-B p56, and β-actin, respectively.

Flow Cytometery
The fluorescein isothiocyanate (FITC)-conjugated primary antibody against ZO-1 and nonconjugated primary antibody against claudin-1 (1:200) were used to detect protein levels, respectively, analyzed by flow cytometry. The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate the production of intracellular superoxide anion. In the presence of superoxide anion, DHE is oxidized to ethidium bromide (EtBr), which is trapped by intercalating with the DNA and emits red fluorescence. Thus, the amount of EtBr correlates well with the level of cellular superoxide anion.

Bio-Plex Luminux Assay
Bio-Plex phosphoprotein and total target assay kits were used on a Luminex multiplex system (Bio-Rad) following the manufacturer’s instructions. The kits can detect the amount of phosphorylated and total JNK and IB in a small amount of cell lysates. Results were presented as the ratio of phosphorylated versus total target proteins.

Statistical Analysis
Data were expressed as the mean±SD. Comparisons were made using the Student t test. A probability value <0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
STC1 Specifically Blocks TNF-–Induced Endothelial Monolayer Permeability in HCAECs
To determine whether STC1 could protect endothelial cells from cytokine-mediated effects, STC1 was added to TNF-–treated HCAECs and endothelial permeability was analyzed using the Transwell system and Texas-Red-labeled dextran tracer. As shown in Figure 1A and 1B, treatment of HCAEC monolayer with TNF- (2 ng/mL) for 24 hours significantly increased endothelial permeability by 55% as compared with untreated cells (n=4, P<0.05). Within the same system, cotreatment with TNF- and STC1 (6.25, 12.5, 25, 50, or 100 ng/mL) blocked TNF-–induced increase in permeability of HCAECs, in a concentration-dependent manner, with an IC50 of 12.5 ng/mL STC1, and complete inhibition of TNF-–induced changes in endothelial permeability by STC1 at a concentration greater than 25 ng/mL (n=4, P<0.05). In the control experiment, STC1 alone had no effects on endothelial permeability. Thus, STC1 effectively blocks TNF-–induced endothelial permeability.

View larger version (17K): [in this window] [in a new window]   Figure 1. Effects of STC1 on TNF-–induced monolayer permeability in HCAECs. A and B, Concentration-dependent study. HCAECs were treated with TNF- and increasing concentrations of STC1 for 24 hours. Endothelial permeability was analyzed using the Transwell system and Texas-Red-labeled dextran tracer. C, Time course study. D, Antibody blocking study. n=4. *P<0.05.

As a time course study, cotreatment of HCAECs with STC1 (50 ng/mL) and TNF- effectively blocked TNF-–induced increase in permeability for extended periods (12, 24, and 48 hours; Figure 1C). In a separate experiment, we examined the effect of STC1 (50 ng/mL) on TNF-–induced changes in endothelial permeability at earlier time points (30, 90, 120, and 240 minutes). As shown in supplemental Figure I, TNF- (2 ng/mL) did not increase endothelial permeability at 30, 90, and 120 minutes, while it significantly increased endothelial permeability by 66% at 240 minutes compared with untreated controls (n=4, P<0.05). STC1 could significantly block TNF-–induced endothelial permeability by 27% at 240 minutes compared with TNF-–treated groups (n=4, P<0.05). Thus, the effects of STC1 on endothelium permeability occur within 3 hours and precede TNF-–induced changes.

To further demonstrate the specificity of STC1 effect on HCAECs, a specific neutralizing antibody against STC1 was used. Indeed, anti-STC1 antibody could block the effects of STC1 on TNF-–treated HCAECs in a concentration-dependent manner (Figure 1D, n=4, P<0.05). Isotype control IgG or heat-inactivated anti-STC1 antibody had no effect on TNF-–induced rise in permeability of HCAEC monolayer.

STC1 Does Not Block VEGF-Induced Endothelial Monolayer Permeability in HCAECs
Because TNF- and VEGF increase endothelial permeability through different mechanisms,^24,25 we determined whether STC1 could affect VEGF-induced changes in endothelial permeability. Treatment of HCAECs with VEGF (20 ng/mL) for 24 hours significantly increased monolayer cell permeability compared with vehicle-treated controls, and addition of STC1 at concentrations that effectively blocked TNF-–induced increase in endothelial permeability (25, 50, and 100 ng/mL) failed to block VEGF-induced increase in HCAEC monolayer permeability (Figure 2A). These data indicate that STC1 specifically inhibits TNF-–induced changes in endothelial permeability, but not VEGF-induced signaling pathways regulating endothelial permeability.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effects of STC1 and nifedipine on monolayer permeability in HCAECs. A, VEGF-induced monolayer permeability. HCAECs were treated with VEGF (20 ng/mL) with or without STC1 for 24 hours. B, Effect of nifedipine. HCAECs were treated with TNF- (2 ng/mL) with or without nifedipine for 24 hours. n=4. *P<0.05.

Calcium Channel Blocker Nifedinine Does Not Block TNF-–Induced HCAEC Monolayer Permeability
Because STC1 is an L-type calcium channel blocker,^1,2 we determined whether L-type calcium channels could be involved in TNF-–induced changes in endothelial permeability. As shown above, TNF- induced a significant increase in HCAEC monolayer permeability. However, increasing concentrations of an L-type calcium channel blocker nifedipine (0.1, 1, 10, and 50 nmol/L) had no effects on TNF-–induced permeability increase in HCAECs (Figure 2B). These data indicate that TNF-–induced changes in endothelial permeability are not L-type calcium channel-mediated, and that STC1 modulates endothelial permeability through a mechanism which is L-type calcium channel-independent.

STC1 Blocks TNF-–Induced Downregulation of ZO-1 and Claudin-1 in HCAECs
Beause the mechanism of TNF-–induced endothelial permeability involves the destruction of endothelial junction structures,^26,27 we determined whether STC1 could affect the expression of junction molecules in TNF-–treated cells. The mRNA and protein levels of ZO-1 and claudin-1 were analyzed by real-time PCR, Western blot, and flow cytometery, respectively. As shown in Figure 3A, TNF- (2 ng/mL) treatment for 24 hours significantly reduced mRNA levels of ZO-1(34%) and claudin-1(57%) compared with untreated groups (n=4, P<0.05), and STC1 (25, 50 and 100 ng/mL) significantly blocked TNF-–induced decrease in ZO-1 and claudin-1 mRNA levels compared with TNF-–treated groups (n=4, P<0.05). Protein levels of ZO-1 and claudin-1 were also decreased substantially in TNF-–treated cells, whereas STC1 effectively blocked the effect of TNF- on ZO-1 and claudin-1 protein levels in HCAECs (Figure 3B and supplemental Figure II).

View larger version (24K): [in this window] [in a new window]   Figure 3. Effects of STC1 on TNF-–induced downregulation of ZO-1 and claudin-1 in HCAECs. HCAECs were treated with TNF- (2 ng/mL) with or without STC1 for 24 hours. A, The mRNA levels of ZO-1 and claudin-1 were analyzed by real-time PCR. n=4. *P<0.05. B, The protein levels of ZO-1 and claudin-1 analyzed with Western blot.

STC1 Blocks TNF-–Induced Superoxide Anion Production in HCAECs
Because oxidative stress is involved in TNF-–induced increase in endothelial permeability, we determined whether STC1 could inhibit TNF-–induced superoxide anion production in HCAECs. Cellular superoxide anion levels were determined by fluorescent dye DHE staining and flow cytometery analysis. There were 30% of DHE-positive cells in untreated controls, compared with 63% in TNF-–treated cells (Figure 4). TNF- and STC1 cotreatment decreased DHE staining from 63% to 43%. These data demonstrate that STC1 can effectively inhibit TNF-–induced oxidative stress in HCAECs.

View larger version (8K): [in this window] [in a new window]   Figure 4. Effects of STC1 on TNF-–induced superoxide anion production in HCAECs. HCAECs were treated with TNF- (2 ng/mL) with or without STC1 (50 ng/mL) for 24 hours. Cellular superoxide anion levels were determined by fluorescent dye DHE staining and flow cytometery analysis.

STC1 Partially Blocks TNF-–Induced Activation of JNK and NF-B in HCAECs
Because the effect of TNF- is mediated through signal transduction pathways including MAPK JNK and transcription factor NF-B,²⁸ we determined whether STC1 could inhibit TNF-–induced activation of JNK and NF-B in HCAECs. JNK activation was directly measured as phospho-JNK/total JNK by Bio-Plex luminex immunoassay. NF-B activation was studied indirectly by measuring the activity of IB, an inhibitor NF-B, determined as phospho-IB/total IB.²⁹ HCAECs were treated with TNF- in the presence or absence of STC1 for increasing times (from 1 to 120 minutes). TNF- treatment substantially increased JNK phosphoarylation, which peaked at 90 minutes (Figure 5A), and IB phosphorylation, which peaked at 20 minutes (Figure 5B). Addition of STC1 partially inhibited TNF-–induced phosphorylation of both JNK and IB in HCAECs. Furthermore, we performed Western blot analysis to determine cytoplasmic IB phosphorylation and nuclear NF-B levels. As shown in Figure 5C, TNF- (2 ng/mL) treatment for 20 minutes increased cytoplasmic IB phosphorylation, whereas STC1 (50 ng/mL), JNK inhibitor SP600125 (10 µmol/L), or SOD mimetic MnTBAP (3 µmol/L) effectively blocked TNF- induced cytoplasmic IB phosphorylation compared with TNF-–treated groups. Concomitantly, TNF- treatment for 20 minutes, but not 1 minute, increased nuclear NF-B (p65) levels, whereas STC1, SP600125, or MnTBAP effectively blocked TNF-–induced increase in nuclear NF-B (p65) levels compared with TNF-–treated groups. These data suggest that oxidative stress and JNK activation are responsible for TNF-–induced NF-B activation. STC1 may inhibit TNF-–induced activation of NF-B through suppression of superoxide generation and inhibition of JNK.

View larger version (20K): [in this window] [in a new window]   Figure 5. Effects of STC1 on TNF-–induced phosphorylation of JNK and IB and nuclear NF-B levels in HCAECs. HCAECs were treated with TNF- with or without STC1, JNK inhibitor (SP600125), or antioxidant (MnTBAP) for different time points. A and B, The phosphorylation of JNK and IB was determined by Bio-Plex luminex immunoassay. C, Cytoplasmic IB and nuclear NF-B levels were determined with Western blot analysis. D, Effects of JNK inhibitor and antioxidant on TNF-–induced monolayer permeability. n=4, *P<0.05.

JNK Inhibitor and Antioxidant Block TNF-–Induced HCAEC Monolayer Permeability
We performed experiments of endothelial permeability with TNF-, STC1, JNK inhibitor (SP600125), and MnTBAP. As shown in Figure 5D, SP600125 (10 µmol/L) or MnTBAP (3 µmol/L) significantly blocked TNF-–induced endothelial permeability compared with TNF- alone–treated cells (n=4, P<0.05). These data suggest oxidative stress and JNK activation are directly responsible for the TNF-–induced endothelial permeability. STC1 may inhibit TNF-–induced increase in endothelial permeability through suppression of superoxide generation and inhibition of JNK.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well known that TNF- can induce endothelial permeability in many inflammatory conditions including cardiovascular disease and tumor progression. However, it is largely unknown whether naturally occurring molecules could inhibit the effects of TNF- during inflammation. In the present study, we demonstrate a novel function of STC1 which can inhibit TNF-–induced increase in endothelial permeability in HCAECs. STC1 specifically inhibits a series of TNF-–induced events in HCAECs including the attenuation of tight junction molecules ZO-1 and claudin-1, overproduction of superoxide anion, and activation of JNK and NF-B in HCAECs.

Endothelial cells form the main physical barrier for blood constituents. They also actively control the extravasation of blood components to the surrounding tissues. In many inflammatory conditions, endothelial permeability increases in response to inflammatory cytokines and growth factors. Several edema-producing agents, including thrombin, VEGF, histamine, IL-1, interferon (INF)-, and TNF- stimulate protein tyrosine phosphorylation and other signaling pathways in endothelial cells to increase permeability.³⁰ Increased endothelial permeability to circulating macromolecules is an early and important event in the pathogenesis of atherosclerosis.³¹ In the present study, we have confirmed that TNF- significantly increases HCAEC monolayer permeability using the Transwell system, a well characterized and accepted simple and reliable model for in vitro endothelial cell permeability assay. More importantly, STC1 can effectively inhibit the increase in endothelial permeability in response to TNF- treatment in the same model. Inhibitory action of STC1 is very potent because a very small concentration of STC1 (25 ng/mL) could completely block the effect of TNF- in HCAECs. These novel findings identify a new role for STC1 in stabilizing the endothelial barrier during inflammation. These discoveries are complementary to recent observations suggesting that STC1 inhibits transendothelial migration of macrophages and T-lymphocytes through quiescent or IL-1β–activated human umbilical vein endothelial cells (HUVECs) in vitro.¹¹ In addition, STC1 regulates gene expression in cultured endothelial cells, and is detected on the apical surface of endothelial cells in vivo. We previously showed increased expression of STC1 in cardiac vessels and cardiomyocytes of patients with dilated cardiomyopathy.¹⁶ The increase in STC1 protein expression in blood vessels of the failing heart suggested an important role for this protein in heart failure. Heart failure is characterized by increased oxidative stress¹⁸ and upregulated expression of inflammatory cytokines such as TNF-, IL-1, and IL-6.¹⁹ Upregulated expression of TNF- and other cytokines in the failing heart is expected to induce ROS and activate JNK and NF-kB in endothelial cells, and thus affect endothelial barrier. Our studies strongly suggest that STC1 plays a critical role in regulating endothelial function under normal conditions and during inflammation including heart failure and atherosclerosis, and could advance our understanding of anti-inflammation pathways, which may have therapeutic value in many inflammatory diseases.

Specificity of the inhibitory effect of STC1 on TNF-–induced endothelial permeability is carefully addressed in the present study. Effects of STC1 are concentation- and time-dependent. Specific antibodies against STC1, but not other control antibodies, could effectively block STC1 effect on HCAECs. Although both TNF- and VEGF can induce endothelial permeability in HCAECs, they act through different receptors and signal pathways.^24,25 To determine whether STC1 could be a specific inhibitor for TNF-, we performed additional experiments with STC1 and VEGF using the Transwell system. Interestingly, STC1 fails to inhibit VEGF-induced increase in monolayer permeability in HCAECs, indicating that the inhibitory effect of STC1 is specific to TNF- and possibly other cytokines. Are the effects of STC1 on endothelial permeability related to changes in calcium homeostasis? Recent data from our laboratory suggest that STC1 inhibits L-type calcium channel in cardiomyocytes,¹⁶ and diminishes intracellular calcium in cultured murine macrophages.¹⁷ Hence, we examined whether STC1 could modulate the effect of TNF- through inhibition of calcium channels. Notably, increasing concentrations of the L-type calcium channel inhibitor nifedinine failed to block TNF-–induced increase in endothelial permeability in HCAECs. These important data indicate that the inhibitory effects of STC1 on TNF-–induced changes in endothelial permeability are independent of L-type calcium channels. Furthermore, we have also shown STC1 to regulate the expression of many genes in human endothelial cells.¹¹ Collectively, these data indicate that STC1 plays a protective role in regulating endothelial cell functions.

Endothelial tight junctions consist of claudins, occludins, and junctional adhesion molecules that are anchored in the membranes of two adjacent cells. Tight junction membrane proteins interact with scaffold proteins (eg, ZO-1) to connect them with various signal transduction and transcriptional pathways involved in the regulation of tight junction function. It is well documented that TNF- can downregulate several tight junction molecules.³² In the present study, we have confirmed that TNF- significantly decreased the mRNA and protein levels of ZO-1 and claudin-1 in HCAECs, which could contribute to the increase in endothelial permeability. Importantly, STC1 effectively blocks TNF-–induced downregulation of ZO-1 and claudin-1 at both mRNA and protein levels.

Oxidative stress is an important factor which can increase endothelial permeability. ROS such as superoxide anion (O₂·^–) contribute to endothelial barrier dysfunction in response to TNF-.³³ ROS are known to quench NO.³⁴ Thus, inhibition of NO pathway can potentiate agonist-induced increases in vascular permeability or increase basal microvascular permeability.³⁵ ROS can directly activate MAPKs and transcription factor NF-B, which regulate the expression of many genes.³⁶ In the present study, we have demonstrated that TNF- significantly increases the production of superoxide anion in HCAECs, whereas STC1 effectively blocks this effect of TNF-. These data are important and could explain the molecular mechanisms of STC1 inhibitory action on TNF-–induced endothelial dysfunction. Indeed, STC1 significantly inhibits TNF-–induced activation of JNK and NF-B in HCAECs, thereby regulating the expression of ZO-1 and claudin-1. Accordingly, JNK inhibitor and antioxidant can effectively block TNF-–induced NK-B activation and endothelial permeability in HCAECs.

TNF- is expressed as 24-kDa membrane–bound protein that is proteolytically cleaved into its 17-kDa soluble form.³⁷ TNF- in its soluble trimerized form binds to 2 receptors, TNFR1 (p55) and TNFR2 (p75). Human endothelial cells express both of these receptors.³⁸ Binding of TNF- to TNFR1 or TNFR2 induces receptor oligomerization and recruitment of several downstream adaptor and signaling proteins to their cytoplasmicdomains.³⁹ It is not clear whether STC1 could act on the TNF- receptors or upstream signaling molecules to block TNF- activities. Further investigation in this regard is warranted.

In summary, human STC1 has diverse roles in developmental, physiological, and pathological conditions. In the present study, we demonstrate, for the first time, that STC1 can counteract the effect of TNF- on endothelial permeability. STC1 inhibits TNF-–induced signaling pathways including oxidative stress and the activation of MAPK, JNK, and transcriptional factor NF-B. This action of STC1 does not appear to be mediated through changes in L-type calcium channel conductance in human endothelial cells. This study suggests that STC1 is actively involved in the inflammation process as an effective inhibitor.

	Acknowledgments

 
Sources of Funding

This work is partially supported by research grants from the National Institutes of Health (Chen: HL65916, HL72716, and EB-002436; Yao: DE15543; and Sheikh-Hamad: O’Brien Kidney Center grant DK064233–01 and DK062828).

Disclosures

None.

	Footnotes

 
Original received May 7, 2007; final version accepted February 12, 2008.

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