Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

Abstract

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-κB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-κB and Sp1 to bind to their binding sites. Knock-down of either NF-κB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-κB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function.

Podocyte Injury and Albuminuria in Mice with Podocyte-Specific Overexpression of the Ste20-Like Kinase, SLK

“…The mice showed a decreased intestinal barrier function. 46 We have previously demonstrated that SLK expression and activity increase in the developing kidney and in kidneys subjected to ischemia-reperfusion injury. 7 The upregulation of expression may then favor homodimerization and phosphorylation, and possibly reversal of auto-inhibition, together leading to an increase in kinase activity.…”

“…46 Cultured intestinal epithelial cells stably transfected with SPAK showed increased permeability. Transgenic mice that expressed a constitutively active form of SPAK under the control of the villin gene locus control region, which can target SPAK expression only in intestinal epithelial cells were generated to study intestinal barrier function in vivo.…”

Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

“…Intestinal permeability was assessed in vivo (51). In brief, Pggt-Iβ TiΔIEC and control mice were deprived of food and water for 4 hours.…”

Adenosine 2B Receptor Expression Is Post-transcriptionally Regulated by MicroRNA

“…Nuclear Run-on Assay-Nuclear run-on assay was performed according to the protocol of Dr. Yan (14). Briefly, nuclei were isolated from T84 cells that were untreated or treated with 10 ng/ml TNF-␣ for 2-4 h. Cells (5 ϫ 10 7 ) were pelleted in a test tube and washed twice with ice-cold phosphate-buffered saline.…”