Molecular characterization of mouse lens epithelial cell lines and their suitability to study RNA granules and cataract associated genes

Abstract

The discovery of cytosolic RNA granule (RG) component proteins associated with human cataract has initiated investigations on post-transcriptional mechanisms of gene expression control in the lens. Application of established mouse lens epithelial cell lines (LECs) can provide rapid insights on RG function in lens cells, especially because mouse mutants in several RG components are not available. However, although these LECs represent potential reagents for such analyses, they are uncharacterized for lens gene expression or RG formation. Therefore, a detailed molecular and cellular characterization of three permanent mouse LECs 17EM15, 21EM15 and αTN4 is performed in this study. Comparative analysis between microarray gene expression datasets on LEC 21EM15 and iSyTE lens tissue demonstrates that 30% of top 200 iSyTE identified lens-enriched genes are expressed in these cells. Majority of these candidates are independently validated to either have lens expression, function or linkage to cataract. Moreover, analysis of microarray data with genes described in Cat-Map, an online database of cataract associated genes and loci, demonstrates that 131 genes linked to cataract loci are expressed in 21EM15 cells. Furthermore, gene expression in LECs is compared to isolated lens epithelium or fiber cells by qRT-PCR and by comparative analyses with publically available epithelium or fiber-specific microarray and RNA-seq (sequencing) datasets. Expression of select candidate genes was validated by regular and real-time quantitative RT-PCR. Expression of lens epithelium-enriched genes Foxe3, Pax6, Anxa4 and Mcm4 is up-regulated in LEC lines, compared to isolated lens fiber cells. Moreover, similar to isolated lens epithelium, all three LECs exhibit down-regulation of fiber cell-expressed genes Crybb1, Mip and Prox1 when compared to fiber cells. These data indicate that the LEC lines exhibit greater similarity to lens epithelium than to fiber cells. Compared to non-lens cell line NIH3T3, LECs exhibit significantly enriched expression of transcription factors with important function in the lens, namely Pax6, Foxe3 and Prox1. In addition to these genes, all three LECs also express key lens- and cataract-associated genes, namely Dkk3, Epha2, Hsf4, Jag1, Mab21l1, Meis1, Pknox1, Pou2f1, Sfrp1, Sparc, Tdrd7 and Trpm3. Additionally, 21EM15 microarrays indicate expression of Chmp4b, Cryab and Tcfap2a among others important genes. Immunostaining with makers for Processing bodies (P-bodies) and Stress granules (SGs) demonstrates that these classes of RGs are robustly expressed in all three LECs. Moreover, under conditions of stress, 17EM15 and αTN4 exhibit significantly higher numbers of P-bodies and SGs compared to NIH3T3 cells. In sum, these data indicate that mouse LECs 21EM15, 17EM15 and αTN4 express key lens or cataract genes, are similar to lens epithelium than fiber cells, and exhibit high levels of P-bodies and SGs, indicating their suitability for investigating gene expression control and RG function in lens-derived cells.

Systems biology of lens development: A paradigm for disease gene discovery in the eye

“…The recent molecular characterization of mouse lens epithelial cell lines (Terrell et al, 2015) and the establishment of protocols for iPSc-based derivation of lens cells (Anchan et al, 2014; Yang et al, 2010) may facilitate their use in ChIP assays, addressing this problem. Modified assays such as “recovery via protection” (RP)-ChIP-seq and favored amplification RP-ChIP-seq, have recently been developed to capture low-abundance chromatin from a single lens, or from a few hundred cells (~500) (Zheng et al, 2015).…”

“…(Audette et al, 2016; Manthey et al, 2014a; Wolf et al, 2013b). Further, it has impacted the characterization of lens cell lines (Terrell et al, 2015) and establishment of protocols for processing lens microarray and RNA-seq data (Anand et al, 2015; Manthey et al, 2014b). …”

Deficiency of the RNA binding protein caprin2 causes lens defects and features of peters anomaly

“…This finding has initiated the investigation of other posttranscriptional regulatory molecules in eye development (Lachke and Maas, ). Here, we used a systems approach termed iSyTE ( i ntegrated Sy stems T ool for E ye gene discovery) that has previously led to identification and characterization of several important genes that function in the lens (Kasaikina et al, ; Lachke et al, a,b; Wolf et al, ; Manthey et al, ,b; Agrawal et al, ; Terrell et al, ) to identify a second RBP/RG component Caprin2 (Cytoplasmic activation‐ and proliferation‐associated protein 2; also known as RNA granule protein 140, RNG140; C1q Domain‐containing protein 1, C1qdc1; EEG1) as a candidate that likely functions in lens development. Caprin family proteins are conserved among metazoans, and human CAPRIN2 protein shares 51% identity and 73% similarity with Drosophila CAPR across a conserved region termed Homology Region 1 (Papoulas et al, ).…”

TRPM3_miR-204: a complex locus for eye development and disease

“…In mouse anterior eye tissues, Trpm3 transcripts are expressed in ciliary body and lens epithelium [93,146,147] and in mouse lens epithelial cell lines [148,149]. RNA sequencing established that Trpm3 transcripts were enriched in the mouse lens during embryonic development (E10.5-E16.5) [150].…”