Vil-Cre specific Slfn3KO mice exhibit sex-specific differences in lung, stomach, cecum, kidney, and proximal colon differentiation markers and Slfn family members expression levels

The Schlafen (Slfn) family proteins are critical regulators of cell proliferation, induction of immune responses, differentiation, self-restoration, and cell cycle progression. Rodent Slfn3 and human ortholog SLFN12 are critical in the regulation of intestinal epithelial differentiation. Following previous work utilizing Vil-Cre epithelial-specific Slfn3 knockout (VC-Slfn3KO) mice to evaluate Slfn3's role in small intestinal epithelial differentiation, we sought to characterize and distinguish the effects of Slfn3 loss on Slfn family member mRNA expression and differentiation markers for other epithelial cells in the lung, stomach, cecum, and proximal colon. Quantitative PCR analysis of Slfn1, 2, 4, 5, 8, and 9 and multiple differentiation markers were evaluated. We observed gender-specific effects with the loss of Slfn3 on the other Slfn family members and epithelial differentiation markers expression. Lung Slfn4 and 5 were increased only in male VC-Slfn3KO while lung Slfn2 and 8 were decreased only in female VC-Slfn3KO compared to controls. Slfn1, 2, 4, and 9 were increased in the gastric mucosa of male VC-Slfn3KO mice compared to controls. Slfn5 was reduced in female VC-Slfn3KO proximal colonic mucosa compared to controls. Lung AT1 cell differentiation marker Hopx mRNA expression was decreased and Ager was increased in VC-Slfn3KO male mice compared to controls. Lung AT2 differentiation markers and surfactant genes Sftpc and Sftpd were decreased in male VC-Slfn3KO mice. Stomach transcription factors, Lgr5 and Notch1 were increased in male VC-Slfn3KO. Tff1 secretory protein gene was decreased in female VC-Slfn3KO mice. Sucrase isomaltase was greatly increased in male VC-Slfn3KO mice in both cecal and proximal colonic mucosa, but glucose transporter Glut2 was decreased only in the cecum of female VC-Slfn3KO. The changes induced by VC-Slfn3KO in the expression of epithelial differentiation markers and other Schlafen proteins in various target tissues, indicate a complex regulation of gene expression that is sex-dependent

RNA Sequencing of Intestinal Enterocytes Pre- and Post-Roux-en-Y Gastric Bypass Reveals Alteration in Gene Expression Related to Enterocyte Differentiation, Restitution, and Obesity with Regulation by Schlafen 12

Background: The intestinal lining renews itself in a programmed fashion that can be affected by adaptation to surgical procedures such as gastric bypass. Methods: To assess adaptive mechanisms in the human intestine after Roux-en-Y gastric bypass (RYGB), we biopsied proximal jejunum at the anastomotic site during surgery to establish a baseline and endoscopically re-biopsied the same area 6–9 months after bypass for comparison. Laser microdissection was performed on pre- and post-RYGB biopsies to isolate enterocytes for RNA sequencing. Results: RNA sequencing suggested significant decreases in gene expression associated with G2/M DNA damage checkpoint regulation of the cell cycle pathway, and significant increases in gene expression associated with the CDP-diacylglycerol biosynthesis pathway TCA cycle II pathway, and pyrimidine ribonucleotide salvage pathway after RYGB. Since Schlafen 12 (SLFN12) is reported to influence enterocytic differentiation, we stained mucosa for SLFN12 and observed increased SLFN12 immunoreactivity. We investigated SLFN12 overexpression in HIEC-6 and FHs 74 Int intestinal epithelial cells and observed similar increased expression of the following genes that were also increased after RYGB: HES2, CARD9, SLC19A2, FBXW7, STXBP4, SPARCL1, and UTS. Conclusions: Our data suggest that RYGB promotes SLFN12 protein expression, cellular mechanism and replication pathways, and genes associated with differentiation and restitution (HES2, CARD9, SLC19A2), as well as obesity-related genes (FBXW7, STXBP4, SPARCL1, UTS)