mTor is Involved in the Dexamethasone Induced Expression of β3 Integrin in Human Trabecular Meshwork Cells

Abstract

Purpose: αvβ3 integrin signaling is involved in the formation of cross-linked actin networks (CLANs), a structure proposed to play a role in steroid induced glaucoma. The purpose of this study was to determine the effect of dexamethasone (DEX) treatment on the expression of β3 integrin in human trabecular meshwork (HTM) cells.

Methods: Post-confluent HTM cells were treated with either 500nM DEX or 0.1% EtOH for 0-6 days. Western blot analysis and FACS were used to determine changes in protein expression. Changes in mRNA expression were determined using qPCR in the absence or presence of 5μg/ml actinomycin D or 25μg/ml cycloheximide. The glucocorticoid inhibitor RU486 was used to determine if changes in mRNA levels were due to direct glucocorticoid receptor activation. The immunosuppressant rapamycin was used to determine if mTOR was involved. FKBP5 (positive control), and β1integrin subunit (housekeeping gene) were used as controls.

Results: Protein expression of the β3 integrin subunit increased starting after 2 days of DEX treatment and remained high as long as DEX was present and for at least 14 days following the removal of DEX. FACS analysis showed that DEX increased β3 integrin activation at the cell surface and it remained activated for at least 7 days after removal of DEX. By qPCR, DEX treatment of HTM cells induced a 6.2-fold increase (p<0.4) in β3 integrin mRNA after 2 days compared to the EtOH control and remained elevated for 6 days of treatment. FKBP5 mRNA and protein levels also increased in response to DEX. mRNA increased by 7.3-fold (p<0.4) after 1 day of DEX compared to EtOH. In contrast, changes in β1 integrin mRNA and protein levels were not seen. Interestingly, just 1 day of DEX treatment was enough to increase the level of β3 integrin mRNA for 4 days in the absence of DEX. The DEX-induced up-regulation of the β3 integrin mRNA was due in part to an increase in the half-life of β3 integrin mRNA to 60.7 hrs from 22.5 hrs in control cultures (p<0.05) and was dependent on de novo protein synthesis. Treatment with RU486 and rapamycin inhibited the DEX response.

Conclusions: DEX induced increase in β3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvβ3 integrin and is dependent on de novo protein synthesis of an activation factor. This factor may be part of the mTOR pathway.

Keywords: 735 trabecular meshwork • 738 transcription • 487 corticosteroids