Abstract
Purpose :
Elevated intraocular pressure (IOP) is a primary risk factor in the development and progression of glaucoma. The trabecular meshwork (TM) is responsible for maintaining homeostatic IOP. However, specific molecular regulators of IOP remain elusive. Integrin-α7 (ITGA7) binds laminin and acts as a mechanotransducer in muscle tissue. We previously demonstrated that ITGA7 is upregulated in TM in response to elevated pressure, that the integrin α7-β1 heterodimer is expressed in the TM, and that signaling in response to plating on laminin is primarily through Akt. Here we investigate the expression of the three isoforms of Akt in hTM. We further elucidate the impact of ITGA7 on hTM cell signaling by knocking out ITGA7 and measuring its effect on downstream molecules in response to stretch.
Methods :
Knockout of ITGA7 in hTERT-GFP immortalized (CL-27) and primary (2020-1183) hTM cells was achieved via lentiviral CRISPR. Percent knockout was determined using Western blotting post puromycin selection. hTM cells were plated on laminin coated Flexcell plates and stretched for 5, 15 or 30 minutes (n = 3 replicates). Protein expression levels and phosphorylation activity was determined by Western blot with protein and phospho-specific antibodies to the following downstream molecules: Akt, p38, JNK, Erk, mTOR, p70S6K, GSK3b, BAD, and 4EBP1.
Results :
Knockout in the immortalized CL-27 hTM cell line was complete whereas it was approximately 50% in the primary cell strain. In immortalized hTM cells Akt activation in response to stretch on laminin was more pronounced in the absence of ITGA7. This correlated with increased mTOR phosphorylation. The results in primary cells were similar but less pronounced. There was no notable effect of stretch on the phosphorylation of JNK or Erk. We also demonstrate that all three isoforms of Akt are robustly expressed in hTM cells.
Conclusions :
In the absence of ITGA7 Akt activity is increased in hTM cells in response to stretch on laminin. We previously showed that Akt is activated in response to laminin binding, the primary ligand of the integrin α7-β1 heterodimer. Thus an increase in Akt phosphorylation in the absence of ITGA7 is unexpected and suggests a key role in modulating signaling pathways in the TM. Further studies in primary hTM cells will provide insight into whether this effect is shared or specific to immortalized hTM cell lines.
This is a 2021 ARVO Annual Meeting abstract.