W1269 HIV Protease Inhibitors Induce ER Stress and Disrupt Barrier Function in Intestinal Epithelial Cells

Abstract

HIV protease inhibitor (PI)-induced diarrhea is one of the most frequent adverse side effects of these drugs and occurs in 16 to 62% of patients. Recently it has been reported that HIV PIs induce apoptosis and decrease barrier function in intestinal epithelial cells. However, the underlying cellular/molecular mechanisms remain unclear.We have previously demonstrated that HIV PIs induce endoplasmic reticulum (ER) stress, activate the unfolded protein response (UPR), and promote apoptosis in macrophages and hepatocytes. In the present study, we examined whether HIV PI-induced ER stress also plays a role in HIV PI-induced apoptosis and disruption of barrier function in intestinal epithelial cells. Methods: In Vitro studies were conducted in IEC6 cells (normal rat intestinal epithelial cells), differentiated IEC6 (stable Cdx2-transfected, IEC6-Cdx2L1), and stable SEAP-transfected IEC6 cells. HIV PIinduced ER stress was measured by decrease of SEAP activity. The mRNA and protein expression levels of ER stress-related genes were determined by real-time RT-PCR and western blot analysis, respectively. The paracellular permeability was measured by paracellular tracer flux assay and membrane-impermeable molecule, [14C]-mannitol, served as tracer. Lentiviral CHOP siRNA was used to knock down CHOP expression in IEC6 cells. Both C57/B6 wild type and CHOP knock out mice were used in In Vivo studies. Mice were gavaged with individual HIV PI (50 mg/kg) for three weeks. The pathological changes in intestine and colon were determined by histological evaluation. Results: Both ritonavir and lopinavir significantly induced ER stress, increased the expression of GRP78, CHOP, XBP-1 and ATF4, and promoted apoptosis in IEC6 and IEC6-Cdx2L1 cells. Paracellular permeability was increased by ~ 2.5 to 5 fold after cells were treated with 15 μM of ritonavir or lopinavir for 24 h. In contrast, amprenavir neither induced ER stress, nor increased paracellular permeability. Down-regulation of CHOP expression in IEC6 cells reduced HIV PI-induced apoptosis and disruption of barrier function. In Vivo studies demonstrated that ritonavir and lopinavir also significantly induced CHOP, XBP-1 and ATF4 expression in the intestine and colon of wild type mice. Furthermore, histological assessment indicated that ritonavir and lopinavir significantly induced intestinal epithelial damage in wild type mice, but had less effect in CHOP knock out mice. Conclusions: These findings indicate that HIV PI-induced ER stress and subsequent activation of the UPR represents an important cellular/molecular mechanism underlying HIV PI-induced disruption of intestinal barrier function.