ABSTRACT: Abstract CRTC3 is a member of cAMP-responsive element-binding protein (CREB)-regulated transcription coactivators (CRTCs) that plays important roles in regulating CREB-target gene expression and energy metabolism. However, there were nearly no reports about the porcine CRTC3 (pCRTC3). In the current study, we cloned the full length cDNA of pCRTC3 gene and examine its expression pattern and function in intestinal epithelial cells. The full length cDNA sequence of pCRTC3 was 2173 bp (GenBank accession no. MF964215), with a 1,860-bp open reading frame encoding a 620-AA protein. Comparison of the deduced AA sequence with different species including human, mouse, rat, papio, cattle and rabbit showed 89 to 91.9% similarity. The pCRTC3 was highly expressed in small intestine and spleen, to a lesser degree in lung, liver and adipose tissue, and was expressed at a low but detectable level in skeletal muscle, kidney and heart. In addition, high protein levels of pCRTC3 were found in IPEC-J2 cells, in which pCRTC3 was mainly localized in cytoplasm. Furthermore, we constructed pCRTC3-sgRNA1-4 expression plasmids to knockdown pCRTC3 in IPEC-J2 cells. Compared with the Lipofectamine 2000 or pX330-Cas9-empty vector control group, the expression of pCRTC3 was obviously decreased by pCRTC3-sgRNA1-4. Notably, knockdown of pCRTC3 significantly decreased the expression of the porcine tight junction related genes including ZO-1, ZO-2, Occludin, and Claudin-1 by 57.88% (P < 0.01), 40.19% (P < 0.01), 51.59% (P < 0.01) and 35.70% (P < 0.05), respectively. Meanwhile, we found oxidative stress treatment affects the expression of CRTC3 in IPEC-J2 cells.Taken together, we first cloned the full-length sequence of pCRTC3 and revealed the tissue-specific expression pattern, localization and function of pCRTC3 in regulating the expression of intestinal tight junction related genes. This study could provide some useful information for understanding the function of CRTC3 in pigs.
