Loss of Adhesion G‐Protein‐Coupled Receptor L2 Expression Impacts Colonic Epithelial Proliferation
Authors: Bucar Edie B. , Carmelo Anna , Thai Megan H. , Frey Mark R. 
Published: May 2022

BackgroundThe role of adhesion G‐protein‐coupled receptor L2 (ADGRL2, a.k.a. Latrophilin‐2) in human health has been understudied despite discoveries that the receptor is required for both heart development and neural synaptogenesis. This project tests the physiological function of ADGRL2 in the colon where our prior findings that ADGRL2 is lost in human colorectal cancer (CRC), downregulated during hyperproliferative recovery from murine DSS colitis, enriched in stem and progenitor cells, and is inversely correlated with growth arrest. This raises the intriguing possibility that ADGRL2 restricts stem cell expansion and therefore downregulation of ADGRL2 is required for stem‐cell driven regeneration and repair.MethodsThe effect of Adgrl2 on cell proliferation was assessed by histological staining for Ki‐67 antigen or for the thymidine analogue EdU (5‐ethynyl‐2’‐deoxyuridine, administered by intraperitoneal injection 2hr prior to harvest) in intestinal‐epithelial specific Adgrl2 knockout mice (Adgrl2 IE KO). Percent positive cells were quantified per crypt and per crypt region (crypt base, mid crypt, upper crypt) in Image J.Results/ConclusionsAdgrl2 IE KO mice have overall reduced numbers of Ki‐67+ cells (8.2% difference, p = 0.0016) in the colonic epithelium. This seems paradoxical given that we hypothesize a tumor suppressor role for ADGRL2. However, this effect is driven by a pronounced decrease in Ki‐67+ cells in the post‐mitotic upper crypt of Adgrl2 IE KO mice (15.2% difference, p = 0.0121) while percentages of Ki‐67+ cells in the more proliferative mid crypt and crypt base were determined to be roughly equal between groups, suggesting that the observed effect is likely not driven by a reduction in proliferating stem or progenitor cells but is perhaps due to increased cell cycle exit or terminal differentiation with Adgrl2 knockout. Given that Ki‐67 antigen is expressed in all active cell cycle phases, we used EdU labeling of S‐phase proliferating cells to further assess the impact of Adgrl2 loss on colonocyte proliferation. Proliferation is not impaired with loss of Adgrl2 and instead showed a trend towards increased EdU positive cells in the crypt base (6.7% increase, p= 0.0907). As expected, overall number of S‐phase cells in the upper crypt compartment was low for both Adgrl2 KO and WT animals, however a significant decrease in EdU positive cells was still detected in Adgrl2 KO mice (2.98% change, p=0.0500). Based on these data, the loss of Adgrl2 does not decrease proliferative capacity of the stem cell or progenitor niche, but does effect the distribution of active cycling or differentiating cells along the crypt axis, likely by promoting cell cycle exit or terminal differentiation. Further studies will investigate this possibility and the role of Adgrl2 loss on cell renewal in the context of disease. These data support our hypothesis that ADGRL2 modulates epithelial regeneration in the colon, potentially as a stem‐cell specific negative regulator. Mechanistic studies on dysregulated growth and repair in the colon may also illuminate targets to prevent colitis‐associated cancer in IBD patients.