Expression of CXCL6 and BBS5 that may be glaucoma relevant genes is regulated by PITX2

Abstract

The transcription factor PITX2 is implicated in glaucoma pathology. In an earlier study we had used microarray analysis to identify genes in the trabecular meshwork (TM) that are affected by knock down of PITX2. Here, those studies were pursued to identify genes that are direct targets of PITX2 and that may be relevant to glaucoma. Initially, bioinformatics tools were used to select among the genes that had been affected by PITX2 knock down those that have PITX2 binding sites and that may be involved in glaucoma related functions. Subsequently, the effect of PITX2 was tested using the dual luciferase assay in four cell cultures including two primary TM cultures co-transfected with vectors containing promoter fragments of six candidate genes upstream of a luciferase gene and a vector that expressed PITX2. Finally, the effect of PITX2 on endogenous expression of two genes was assessed by over expression and knock down of PITX2 in TM cells. Thirty four genes were found to contain PITX2 binding sites in their putative promoter regions, and 16 were found to be associated with TM-specific and/or glaucoma associated functions. Results of dual luciferase assays confirmed that two of six genes tested were directly targeted by PITX2. The two genes were CXCL6 (chemokine (C-X-C motif) ligand 6) and BBS5 (Bardet-Biedl syndrome 5). Over expression and knock down of PITX2 showed that this transcription factor affects endogenous expression of these two genes in TM cells. CXCL6 encodes a pro-inflammatory cytokine, and many studies have suggested that cytokines and other immune system functions are involved in glaucoma pathogenesis. BBS5 is a member of the BBS family of genes that affect ciliary functions, and ciliary bodies in the anterior chamber of the eye produce the aqueous fluid that affects intraocular pressure. Immune related functions and intraocular pressure are both important components of glaucoma pathology. The role of PITX2 in glaucoma may be mediated partly by regulating the expression of CXCL6 and BBS5 and thus affecting immune functions and intraocular pressure.

Genetic association and differential expression of PITX2 with acute appendicitis

“…Further, we speculate that PITX2 may be involved in anti-oxidant or regenerative responses in the appendix. In the heart PITX2 promotes regeneration after injury through anti-oxidant response (Tao et al 2016), and in the eye it may mediate the response to oxidative stress (Paylakhi et al 2011; Strungaru et al 2011), where it targets a pro-inflammatory cytokine (Moazzeni et al 2016) and a gene encoding response to oxidative stress (Strungaru et al 2011). PITX2 also promotes skeletal muscle regeneration through its repression of miR-31 (Vallejo et al 2018).…”

The Primary Cilium as a Therapeutic Target in Ocular Diseases

“…Bardet-Biedl syndrome 5 (BBS5) has also been found to be localized in trabecular meshwork cilia. Microarray analysis and dual luciferase assays confirmed that BBS5 is regulated by the transcription factor paired like homeodomain 2 (PITX2), and the absence of PITX2 not only activates pro-inflammatory cytokines, but also reduces BBS5 to damage cilia ( Moazzeni et al., 2016 ).…”

Insights into the regulatory molecules involved in glaucoma pathogenesis

“…PITX2 regulates expression of additional potentially glaucoma‐relevant genes, including CXCL6 , BBS5 , and ALDH1A1 (Moazzeni, Akbari, Yazdani, & Elahi, 2016; Paylakhi et al, 2011; Paylakhi et al, 2013). CXCL6 has immune functions (Moazzeni et al, 2016; Paylakhi et al, 2011; Paylakhi et al, 2013).…”

“…PITX2 regulates expression of additional potentially glaucoma‐relevant genes, including CXCL6 , BBS5 , and ALDH1A1 (Moazzeni, Akbari, Yazdani, & Elahi, 2016; Paylakhi et al, 2011; Paylakhi et al, 2013). CXCL6 has immune functions (Moazzeni et al, 2016; Paylakhi et al, 2011; Paylakhi et al, 2013). BBS5 may affect functions of ciliary bodies which are the source of aqueous humor in the anterior chamber (Coca‐Prados & Escribano, 2007; Kiel, Hollingsworth, Rao, Chen, & Reitsamer, 2011; Nachury et al, 2007).…”