Supporting data for “Unraveling the function of Lymphocyte Function-Associated Antigen 1 (LFA-1) in the testis”.
In the testes, LFA-1 is mainly expressed in germ cells, with a notable localization at the heads of elongating spermatids during stages V-VIII of the seminiferous cycle, indicating its potential role in mediating germ cell-Sertoli cell adhesion and maintaining germ cell polarity. Previous research demonstrated that testicular LFA-1 does not interact with ICAM-1 and JAM-A, which are well-known LFA-1 binding partners in the immune system. Following immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis using an anti-αL integrin antibody, three novel LFA-1 binding partners were identified: calreticulin (CRT), doppel (Dpl), and 14-3-3θ.
To investigate the role of LFA-1 in germ cells, knockdown cell lines and overexpression vectors for LFA-1, CRT, Dpl, and 14-3-3θ were constructed. Several junctional proteins, including N-cadherin, nectin-2, coxsackie- and adenovirus receptor (CAR), JAM-B, and JAM-C, were found to be regulated by LFA-1. Additionally, CRT, Dpl, and 14-3-3θ differentially modulated the expression of these junctional proteins. A cell adhesion assay revealed that fewer germ cells adhered to Sertoli cells in the absence of LFA-1, CRT, and Dpl. Low-density lipoprotein receptor-related protein 1 (LRP1) was found to interact with and negatively regulate the LFA-1 protein complex. LRP1 knockdown by siRNA and inhibition with its antagonist upregulated junctional proteins, mirroring the effects observed in LFA-1 overexpression. Furthermore, inhibiting LRP1 with its antagonist LRPAP1 enhanced germ-Sertoli cell adhesion, an effect similar to LFA-1 overexpression. A regulatory feedback loop involving LFA-1, 14-3-3θ, and LRP1 was identified, with Dpl acting as a positive regulator of LFA-1. Knockdown of LFA-1 and Dpl reduced the polarized localization of LRP1 and decreased the expression of the cell polarity protein PAR3, suggesting their role in regulating cell polarity. Additionally, FAK, RasGAP, b-Raf, and HSP70 were differentially regulated by the LFA-1 protein complex, indicating their potential involvement in LFA-1-mediated signaling pathways governing cell adhesion and polarity.
