Non-Hematopoietic β-Arrestin1 Confers Protection Against Experimental Colitis

Abstract

β-Arrestins are multifunctional scaffolding proteins that modulate G protein-coupled receptor (GPCR)-dependent and -independent cell signaling pathways in various types of cells. We recently demonstrated that β-arrestin1 (β-arr1) deficiency strikingly attenuates dextran sodium sulfate (DSS)-induced colitis in mice. Since DSS-induced colitis is in part dependent on gut epithelial injury, we examined the role of β-arr1 in intestinal epithelial cells (IECs) using a colon epithelial cell line, SW480 cells. Surprisingly, we found that knockdown of β-arr1 in SW480 cells enhanced epithelial cell death via a caspase-3-dependent process. To understand the in vivo relevance and potential cell type-specific role of β-arr1 in colitis development, we generated bone marrow chimeras with β-arr1 deficiency in either the hematopoietic or non-hematopoietic compartment. Reconstituted chimeric mice were then subjected to DSS-induced colitis. Similar to our previous findings, β-arr1 deficiency in the hematopoietic compartment protected mice from DSS-induced colitis. However, consistent with the role of β-arr1 in epithelial apoptosis in vitro, non-hematopoietic β-arr1 deficiency led to an exacerbated colitis phenotype. To further understand signaling mechanisms, we examined the effect of β-arr1 on TNF-α-mediated NFκB and MAPK pathways. Our results demonstrate that β-arr1 has a critical role in modulating ERK, JNK and p38 MAPK pathways mediated by TNF-α in IECs. Together, our results show that β-arr1-dependent signaling in hematopoietic and non-hematopoietic cells differentially regulates colitis pathogenesis and further demonstrates that β-arr1 in epithelial cells inhibits TNF-α-induced cell death pathways.

Figure 1. Effect of β-arrestin 1 knockdown on intestinal epithelial cell proliferation and apoptosis

Human intestinal epithelial cell line (SW480) was transfected with either control or β-arr1 siRNA for 48 hrs (A). Transfected cells were treated with TNF-α (20 ng/ml) at the indicated times. (B). DNA content is expressed as mean fluorescence intensity. For apoptosis, after transfection with siRNA, SW480 cells were incubated with TNF-α and CHX for the indicated time points, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry. The representative apoptosis pattern is shown and the apoptotic cells (Annexin V positive, PI negative) are indicated as the percentage of cells (C). Quantitative analysis of apoptosis is shown as bar graph (D). (N=5 independent experiments)

Figure 2. Effect of β-arrestin1 knockdown on TNF-α-induced caspase-3 and caspase-8 activation in intestinal epithelial cell line

Control or β-arr1 knockdown cells were treated with TNF-α and CHX for 12, 24, and 36 hours as indicated or left untreated for 36 hours (UT). Whole cell lysates were subjected to Western blotting for caspase-3 (A) and caspase-8. Representative blot is shown on the left and quantitation on the right for caspase-3. Data are mean±SEM. N=3. *p< 0.05, **P<0.001 compared with controls. Effect of the treatments on caspase-8 activation is shown in B. Representative blot is shown in the top and quantitation in the bottom for caspase-8. Data are mean±SEM. N=3.

Figure 3. Increased gross and histopathological disease assessment in BM chimeric mice lacking β-arrestin1 in non-hematopoietic cells after DSS treatment

BM chimeras were subjected to 2% DSS in drinking water as described in the methods. Body weight is shown in A, histopathology is shown in B (slides on the top and quantitation in the bottom). Note that the percent mucosal involvement was compared by Fisher Exact test. P=0.021 for comparison of minimal and moderate damage between WT and non-hematopoietic KO; P<0.0001 for comparison of minimal and severe damage between WT and non-hematopoietic KO. Colon lengths are shown in C. Data are pooled from 2 independent experiments (N=10). *P<0.05, **P<0.01, ****P<0.0001 compared to the corresponding WT treatment.

Figure 4. Effect of β-arrestin1 knockdown on TNF-α-induced reactive oxygen species in intestinal epithelial cell line

Control and β-arr1 knockdown SW480 cells were loaded with 10 μM CM-H₂DCFDA for 30 min. After washing, the cells were treated with TNF-α and CHX for 12 and 24 hours or left untreated (UT) for 24 hours. The fluorescence intensities were analyzed by flow cytometry. Untreated cells (black lines) were used as a negative control. In the top is representative histogram of one set of triplicate experiments. Blue and red lines represent 12 and 24 hours after stimulation, respectively. Quantitation of ROS production is shown as mean fluorescence intensity in the bottom (N=3 independent experiments).

Figure 5. TNF-α-induced intestinal epithelial cell apoptosis is dependent on β-arrestin1-mediated JNK and caspase-3 signaling pathways

Control and β-arr1 knockdown SW480 cells were treated with TNF-α for 15, 30 and 60 minutes or left untreated (UT) for 60 minutes. Whole cell lysates were subjected to Western blotting for pERK1/2, pJNK, p-p38, pNFκB65. Representative blots are shown in A. Quantitation was done by normalizing phopho-blots with tubulin or ERK2 (B). N=3. *P<0.05, **P<0.01 compared with controls.