Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation

Abstract

Background/aims: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation.

Methods: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells.

Results: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes.

Conclusions: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.

Keywords: Differentiation; Epithelium; Intestine; Schlafen 12; Serpin B12; Signaling; UCHL5; USP14.

Fig. 1.

Duodenal Schlafen 12 is reduced in fasting humans in parallel with enterocyte differentiation marker expression. (a)Quantitative real-time PCR (qRT-PCR) for SLFN12, Sucrase-isomaltase (SI), Villin, DPPIV and GLUT2 from laser-captured epithelium from biopsied duodenal mucosa of control or fasting patients. Expression of SLFN12, SI, Villin, DPPIV and GLUT2 was significantly decreased in fasting patients vs. control (n=12–18,*p<0.05 by two-sided t-test with Bonferroni correction). 18S mRNA served as housekeeping control. (b) Representative SLFN12, SI, and villin stains demonstrate decreased immunoreactivity in fasting vs. control human duodenal biopsies. (Images shown are representative of 6 in each group.) (c) SLFN12 immunostaining with the same antibody for antibody validation. i) represents Caco-2 cells stained with FITC alone as a nuclear counterstain. ii) depicts wild type Caco-2 cells stained with anti-SLFN12 and the FITC counterstain. iii) shows increased SLFN12 immunoreactivity in Caco-2 cells infected with adenovirus encoding SLFN12. iv) represents wild type Caco-2 cells transfected with nontargeting NT1 siRNA, while v) represents paired Caco-2 cells similarly transfected with siRNA to SLFN12, demonstrating decreased immunoreactivity. vi) demonstrates the absence of SLFN12 immunoreactivity in rat IEC-6 cells stained with FITC and antibody to SLFN12.

Fig. 2.

Schlafen 12 alters Caco-2 differentiation markers. (a) Flow cytometry correlating Caco-2 Slfn12 and sucrase-isomaltase (SI) protein. (b) Caco-2 cells co-transfected with human SI-promoter reporter or human SLFN12-promoter reporter constructs without or with empty vector (pEGFP-C1) or SLFN12 constructs (pEGFP-C1-SLFN12). SLFN12 enhances SI-promoter activity (n=6,*p<0.05 vs empty vector alone, left two bars) and SLFN12 promoter activity (n=6,*p<0.05 vs empty vector alone, right two bars). (c) SI expression in Caco-2 cells transduced with empty control virus (Ad-CMV) or SLFN12 adenovirus (Ad-SLFN12) for 72 hours (n=6,*p<0.05 vs empty control virus) Ribosomal Protein Lateral Stalk Subunit P1 [RPLP0] served as a control. (d) SI protein in Caco-2 cells (n=6,*p<0.05 vs empty control virus) with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control. (e,f) siRNA to SLFN12 (siSLFN12) reduces SLFN12 and SI expression vs. non-targeting control siRNA (n=6,*p<0.05 vs siNT1). (g) siSLFN12 decreases Caco-2 SI promoter-driven luciferase activity vs. siNT1 in the presence of empty vector or SI promoter reporter constructs. (n=6,*p<0.05 vs siNT1). (h) siSLFN12 decreases Caco-2 SI protein levels by Western blot vs. siNT1. (n=6,*p<0.05 vs siNT1). (i) Caco-2 cells were transduced with Ad-CMV or Ad-SLFN12 in 4mM hydroxyurea. SLFN12 overexpression stimulates SI expression during complete hydroxyurea proliferative blockade (n=6,*p<0.05 vs empty control virus). (j) SLFN12 overexpression similarly increases SI protein levels after transduction with Ad-SLFN12 in 4 mM hydroxyurea vs. transduction with Ad-CMV. (n=6,*p<0.05 vs empty control virus). All statistics are by two-sided t test with Bonferroni corrections for multiple comparisons.

Fig. 3.

Schlafen 12 acts through SerpinB12. (a) Lysates of Caco-2 cells transfected with empty vector (GST-alone) or SLFN12 (GST-SLFN12) were purified on GST columns. (b) Coprecipitation with antibody to GFP of SerpinB12 protein from Caco-2 cells overexpressing empty vector (pEGFP-C1), SLFN12 (pEGFP-C1-SLFN12), or a SLFN12 point mutant (pEGFP-C1-SLFN12, D233Q). Constructs and lysates were immunoprecipitated with monoclonal anti-GFP and immunoblotted with polyclonal anti-SerpinB12 (n=4,*p<0.05 by ANOVA followed by two-sided t test with Bonferroni correction). (c) Point mutations to the atypical ATP-binding region of SLFN12 prevent induction of sucrase-isomaltase (SI) promoter activity. Caco-2 cells were co-transfected with human SI-promoter reporter constructs without or with empty vector (pEGFP-C1) or SLFN12 wild-type construct (pEGFP-C1-SLFN12) or SLFN12 mutants (p-EGFP-C1-SLFN12 L222A, mutant1; p-EGFP-C1-SLFN12 D233A, mutant2; p-EGFP-C1-SLFN12 D233Q, mutant3; p-EGFP-C1-SLFN12 D233T, mutant4; and p-EGFP-C1-SLFN12 Y236F, mutant5) before luciferase reporter assay (n=6,*p<0.05 by ANOVA followed by two-sided t test with Bonferroni correction). (d) Caco-2 cells were infected with a lentivirus encoding either a V5 tag (as a control) or V5-tagged SLFN12, lysed, and immunoprecipitated with antibody to the V5 tag, prior to immunoblotting for Serpin B12 (n=3, *p<0.05), V5 (not seen in control lanes because the V5 tag alone is too small and runs off the gel), and Serpin B5, which was not observed to coprecipitate although we confirmed that the Serpin B5 antibody could be used for Western blotting in these cells (not shown). (e) Caco-2 cells were transfected with empty vector (pEGFP-C1) or SerpinB12 (pEGFP-C1-SerpinB12) constructs for 72 hours. Lysates were immunoprecipitated with monoclonal anti-SerpinB12 and immunoblotted with polyclonal anti-SLFN12. (f) Flow cytometry correlating endogenous Caco-2 SerpinB12 and SI expression. (g) Caco-2 cells were co-transfected with human SI-promoter reporter construct without or with empty vector (pEGFP-C1) or SerpinB12 (pEGFP-C1-SerpinB12) and luciferase reporter activity was assayed (n=6,*p<0.05). (h) Caco-2 cells were transduced with AAV-SerpinB12 virus or empty control virus (AAV-ZsGreen) and SI mRNA was measured (n=6,*p<0.05). (i) Caco-2 cells were transduced with Ad-CMV or Ad-SerpinB12 and western blot for SI protein was performed (n=6,*p<0.05 vs empty control virus). (j) Caco-2 cells were incubated with siNT or SerpinB12 siRNA (siSerpinB12), transduced with Ad-CMV or Ad-SLFN12 for 72 hours, and SI mRNA or (k) SI protein was measured (n=6, #p<0.05, to respective siNT; *p<0.05 siNT1-Ad-SLFN12 compared to siNT-Ad-V). All statistics are by two-sided t test with Bonferroni correction, with ANOVA performed as indicated.

Fig. 4.

Schlafen 12 acts by modulating deubuiquitylase activity. (a) Caco-2 cells transfected with non-targeting siRNA5 (siNT5) or a combination of both USP14 (siUSP14) and UCHL5 (siUCHL5) siRNA followed by transduction with control (Ad-CMV) or SLFN12 (Ad-SLFN12) viruses for 72 hours, and SI expression was assessed (n=6,*p<0.05). (b,c) Caco-2 cells were transduced with Ad-CMV or Ad-SLFN12 for 72 hours and USP14 and UCHL5 expression was measured (n=6 each,*p<0.05). (d,e,f) Caco-2 cells were transfected with either nontargeting siRNA NT5 or combined siRNA to UCHL5 and USP15 along with either an empty vector adenovirus (Ad-V) or adenovirus encoding SLFN12 (AdSLFN12). Western blots were performed for sucraseisomaltase (d), USP14 (e), and UCHL5 (f). (g,h) Caco-2 cells were transduced with Ad-CMV or Ad-SERPB12 and USP14 and UCHL5 expression measured. (i) Caco-2 cell lysates were immunoprecipitated with monoclonal USP14 or monoclonal SerpinB12 antibodies and immunoblotted with polyclonal SerpinB12 antibody. We immunoprecipitated Serpin B12 directly as a control to confirm the correct apparent molecular weight of the co-precipitating Serpin B12. (j) Incubation with recombinant human Serpin B12 stimulated deubuiquitylase activity vs. USP14 alone (n=4,*p<0.05 to substrate (sub) or SERPB12 alone; # p<0.05 vs. USP14 alone). (k) Incubation with recombinant human Serpin B12 did not affect the UCHL5 deubuiquitylase activity vs. UCHL5 alone (n=4,*p<0.05 to substrate (Sub) or Serpin B12). All statistics are by two-sided t test with Bonferroni corrections for multiple comparisons.

Fig. 5.

Schlafen 12 induces CDX2 transcription factor and its effects are blocked by reducing USP14 and UCHL5 deubiquitylase. (a,b) Caco-2 cells were transduced with Ad-CMV or Ad-SLFN12 for 72 hours and CDX2 or CDX4 protein was measured (n=5 for each, p>0.05). (c) Caco-2 cells were transduced with Ad-CMV or Ad-SLFN12 for 72 hours and CDX2 mRNA was assessed (n=5, p>0.05). (d) Caco-2 cells transfected with non-targeting siRNA5 (siNT5) or a combination of both USP14 (siUSP14) and UCHL5 (siUCHL5) siRNA followed by transduction with Ad-CMV or Ad-SLFN12 for 72 hours, lysis, and western blotting using CDX2 or GAPDH antibodies (n=6,*p<0.05). (e) ≈50% reduction of CDX2 by siRNA (siCDX2), data not shown prevents the induction of sucrase-isomaltase promoter activity by cotransfection with a SLFN12 plasmid (SI-P+SLFN12) in comparison to sucrase-isomaltase promoter activity after cotransfection with an empty vector control plasmid (P). In contrast, SI-P+SLFN12 induces sucrase-isomaltase promoter activity after transfection with non-targeting siRNA (siNT5). (n=6, *p<0.05). All statistics are by two-sided t test with Bonferroni corrections for multiple comparisons.

Fig. 6.

The Schlafen 12-Serpin B12 pathway regulates enterocytic sucrase-isomaltase expression in HIEC-6 cells. Infection with adenovirus expressing either SLFN12 (AdSLFN12) or SERPB12 (AdSERPB12) increases sucrase-isomaltase (SI) and villin 1 (Vil1) mRNA in HIEC-6 cells vs. adenovirus expressing only the CMV promoter (Adv-CMV) (n=6 pooled from 3 separate experiments, *p<0.05).