β1-integrin may regulate Egr1 (early growth response 1) within the lens
University of Delaware
Integrins are heterodimeric transmembrane extracellular matrix receptors that regulate cell-to-cell and cell-to-ECM communication and require an α and a β subunit. The β1-integrin subunit is expressed in both epithelial and fiber cells within the lens, an epithelial tissue important for vision, and also interacts with the largest number of α subunits. Full deletion of β1-integrin from the developing lens causes embryonic lethality at the blastocyst stage. Therefore a conditional knock-out was created, deleting β1-integrin from the lens at embryonic day 12.5 (E12.5). This caused severe disorganization of the lens by E16.5, with the absence of the central epithelium, elongation of the epithelial cells at the transition zone, and highly vacuolated fiber cells. These lens epithelial cells exhibited loss of their normal markers and up-regulation of fiber cell and EMT markers. Analysis of candidate genes based on their known roles in integrin, TGFβ, and MAPK pathways failed to explain the observed phenotype. Hence, I used an unbiased analysis of gene expression changes to identify the likely regulator of the β1-integrin null phenotype. To do so, I chose to use next generation RNA-sequencing (RNA-Seq), a novel technique for whole genome expression profiling in the lens. Although many researchers in the past have used expression microarrays, it has several limitations that include a reliance on existing knowledge about genome sequence, high background, and limited dynamic range. RNA-Seq, on the other hand, has very low background signal, unambiguous mapping capability, and increased sensitivity. This increased sensitivity allows us to sequence past a large bias towards structural proteins in the lens. I chose to sequence lenses at E15.5, one day prior to the onset of the β1MLR10 phenotype, in attempt to obtain gene changes that are proximal to the cause of the fibrotic phenotype observed. With the help of Abby Manthey, I was able to collect wild type C57Bl/6 lenses for RNA-Seq, and compare them to β1MLR10 lens samples. Through this comparison, it was found that over 7,700 genes are expressed in the wild type lens at E15.5. To be able to determine biologically relevant gene changes, we compared two wild type mouse strains: C57Bl/6, an inbred strain, to a mixed background wild type strain. This comparison allowed us to determine that the lens expresses over 7,000 genes over 2 Reads Per Kilobase per Million (RPKM), a measurement of gene transcript level. We also found that only 2%, or 32 genes, of over 2,300 differentially expressed genes between the different strains were above a 2.5 fold change. Therefore any fold change less than this was likely to be due to genetic background strain variability, and not the deletion of β1-integrin from the lens. Therefore, to determine biologically relevant gene changes in the β1MR10 lens, I used the following filtering criteria: mean RPKM ≥ 2 p-value ≤ 0.05, and fold change ≥ 2.5. Using these filtering criteria, I was able to determine that 90 genes are differentially expressed three days after the deletion of β1-integrin and one day prior to the onset of the phenotype. A large subset of these genes are known to play a role in fibrotic responses and include: Egr1, Nab2, αSMA, Mmp14, Anxa2, Plat, Mt1, Thbs1, Stmn1, and Rpl29. Egr1, a transcription factor that is directly regulated by integrin signaling, in turn directly regulates MT1, Nab2, and αSMA in other tissues. Egr1 also has potential to regulate a number of differentially expressed genes within the β1MLR10 lens through EGR1 binding sites (EBS) within their promoter regions. This led me to the conclusion that Egr1 is likely able to regulate the partial fibrotic phenotype observed in the β1 conditional knock out lens. Using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis, I was able to validate the differential expression of Egr1, Nab2, αSMA, Mmp14, Anxa2, Plat and Rpl29 at E15.5 in the β1MLR10 lens. Interestingly, Egr1, Nab2, αSMA, Mmp14, Anxa2, Plat (all genes up-regulated in RNA-Seq) are not only found to be up-regulated at E15.5, but are also up-regulated just one day after the full absence of the β1-integrin protein from the lens. This up-regulation continues to rise through E15.5, and remains high through E16.5. Immunolocalization also showed that Egr1 is increased at the protein level at E15.5 and E16.5. Altogether, this strongly supports Egr1’s role in mediating the partial fibrotic response observed in the β1MLR10 lens. With Egr1’s implication in mediating fibrotic responses in the embryonic lens, it seemed likely that it may be able to play a role in lens diseases such as cataracts and posterior capsular opacification (PCO). Cataract, or the loss of lens transparency, is a major disease of the lens that leads to loss of vision, and is the most common cause of blindness worldwide. Cataracts are commonly treated by extracapsular lens extraction, which involves the surgical removal of the bulk of the lens cells by phacoemulsification, and insertion of a prosthetic lens (or intraocular lens, IOL) into the remaining capsular bag. The remaining lens epithelial cells left on the anterior capsule after surgery proliferate and migrate to accumulate between the capsule and the IOL, where they become fibrotic plaques and cause re-clouding of the lens. This clouding of the lens after cataract surgery is known as posterior capsular opacification (PCO). Studies of PCO models have shown that the fibrotic cells express αSMA, CD44 and other markers of epithelial to mesenchymal transition (EMT). Importantly, EGR1 is able to regulate both αSMA and CD44 in other tissues, and is known to be involved in cataracts induced by selenite in the rat lens. Therefore, I hypothesize that Egr1 may be an early regulator in lens injury responses such as PCO. By using a mouse cataract surgery model, I was able to investigate the expression of Egr1, Nab2, αSMA and CD44 at multiple time points after surgery. Considering Egr1 is an immediate early response gene, I analyzed samples at 3, 6, 12, 24 and 48 hours post-surgery. Preliminary results using qRT-PCR analysis with one biological replicate seemed promising, with a strong up-regulation of Egr1 at 3 hours post-surgery. However, subsequent efforts to support this with three biological replicates were unsuccessful. My efforts to detect changes in Nab2, αSMA and CD44 expression over this time frame were also unsuccessful using qRT-PCR. However, immunolocalization showed up regulation of EGR1 protein just 3 hours after cataract surgery in the proliferating epithelial cells remaining in the lens capsule. Overall, Egr1is likely to play a major role in the embryonic fibrotic phenotype observed in the β1MLR10 lens; up-regulating pro-fibrotic pathways through Nab2, αSMA, Mmp14, Anxa2 and Plat. It may also play an early role in PCO pathogenesis and other lens diseases.
Developmental Biology , Lens , Beta-1-integrin , EGR1