E. Mahdi1, A. Glazier1, N. Malkoff1, J. Xu1, N. Noriega1, C. Short1, M. Fenlon1, K. Wang1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA
Introduction: Biliary atresia (BA), a congenital obstructive cholangiopathy, is the leading cause of end-stage liver failure in kids. We previously showed expansion of Prominin-1 (Prom1)-expressing hepatic progenitor cells (HPCs) within areas of periportal fibrosis in BA, with Prom1 knockout leading to decreased biliary ductular reactions and fibrosis typical of BA. Herein, we hypothesized that Prom1 functionally promotes fibrosis.
Methods: PROM1-enriched Mat1a-/- HPCs, which were treated in vitro ± 5 ng/mL profibrogenic recombinant (r) TGFB ± 0.05 µmol X030 (loss-of-function), which inactivates HSP90 downstream of PROM1, to assess effect on fibrogenic gene expression. A stably transfected Prom1-overexpressing Mat1a HPC clone (gain-of function) was then treated with ± 5 ng/mL rTGFB. Relative gene expression was analyzed using quantitative polymerase chain reaction. PROM1-positive and negative cells were collected separately by fluorescence-activated cell sorting (FACS) to assess relative gene fibrogenic expression.
Results: Following rTGFB treatment, Mat1a cells exhibited increased fibrogenic markers Collagen-1a (Col1α) (43.2±5.7, p<0.0001), a-Smooth muscle actin (aSma) (166.1±20.1, p<0.0001), Integrin-B6 (ItgB6) (4.4±0.4, p<0.0001), and Vimentin (2.3±0.7); increased progenitor markers Sox9 (2.8±0.2, p<0.0001) and Cd49f (1.9±0.2, p<0.0008); and decreased cholangiocyte marker Cytokeratin-19 (Ck19) (0.8±0.08, p<0.0116) expression. X030 co-treatment resulted in decreased Col1a (17.1±2.4, p<0.0001), ItgB6 (2.8±0.1, p<0.0001), Sox9 (4.0±0.1, p<0.0001), Cd49f (4.2±0.2, p<0.0001), and Ck19 (0.70±0.01, p<0.0001), but no change in aSma or Vimentin. Prom1-overexpressing cells exhibited a 13,996 (±1263, p<0.0003) fold increase in Prom1 compared to Mat1a cells. They showed no difference in baseline Col1a, aSMA, Vimentin, CK19, or Sox9 levels, but had decreased TGB6 (0.33±0.04, p<0.0001) expression. Prom1 over-expressed cells treated with TGFB, demonstrated increased Col1a (28.0±9.3, p<0.0006), aSma (35.8±3.7, p<0.0001), Vimentin (1.6±0.2, p<0.027), as well as decreased CK19 (0.8±0.1, p<0.04) compared to control. X030 co-treatment resulted in decreased Col1a (12.1±2.7, p<0.01) and aSma (8.4±2.6, p<0.0001), increased albumin (2.7±0.6, p<0.0197), and no change in Ck19 or Sox9. Compared to PROM1-negative cells, FACS-sorted PROM1-positive cells had no difference in Col1α and ITGB6, decreased CK19 (0.7±0.1, p<0.0001), and increased Vimentin (1.7±0.2, p<0.002) at baseline. After TGFB treatment, PROM1-positive cells had increased Col1α (880.3±230.2, p<0.0001) compared to PROM1 negative cells.
Conclusion: Prominin-1 functionally promotes a profibrogenic response in Mat1a null HPC in vitro.