December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Tunicamycin Induced Unfolded Protein Response (UPR) In Cultured Bovine Trabecular Meshwork (TM) Cells
Author Affiliations & Notes
  • R Mitra
    Indiana University Bloomington IN
    School Optometry
  • M Satpathy
    School of Optometry
    Indiana University Bloomington IN
  • SP Srinivas
    Indiana University Bloomington IN
  • Footnotes
    Commercial Relationships   R. Mitra, None; M. Satpathy, None; S.P. Srinivas, None. Grant Identification: Support: EY11107 (SPS)
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 4086. doi:
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      R Mitra, M Satpathy, SP Srinivas; Tunicamycin Induced Unfolded Protein Response (UPR) In Cultured Bovine Trabecular Meshwork (TM) Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4086.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Expression of mutant MYOCs results in myocilin that is NOT secreted [Jacobson et al., Human Molecular Genetics, 10(2), p 117-125, 2001]. We hypothesize that the resulting accumulation in ER, quality control center along the secretory pathway, leads to «unfolded protein response» (UPR; a form of ER stress) leading to transcriptional activation of ER chaperones and glucose metabolism, and disturbances in cellular Ca2+ homeostasis (Paschen, Cell Calcium, 29:1-11, 2001). In this study, we show our first observations of ER stress response in TM cells through evaluation of expression of stress transducers at the mRNA level and effects on [Ca2+]i homeostasis. Methods: Bovine TM cells were grown to confluence in T-25 flasks and then on glass coverslips for [Ca2+]i measurements. ER stress was induced by exposure to tunicamycin, a nucleoside antibiotic that inhibits N-glycosylation in ER. Expression of CHOP (C/EBP homologous protein-10, also known as GADD153), BiP (also known as GRP78), GRP94, TRP-1, TRP-3, TRP-4, TRP-5, and TRP-6 were followed by semi-quantitative RT-PCR with GADPH as control. Fura-2 was employed to measure changes in [Ca2+]i. Experiments were conducted with HCO3--free Ringers at 37°C. Results: Exposure to tunicamycin at 5 µg/mL for 6 hours induced an increase in expression of CHOP mRNA but not BiP or GRP94. Dexamethasone (100 and 500 nM; exposed for 10 days) also caused elevated mRNA levels for CHOP but not for BiP or GRP94. In BTM cells, TRP-1, TRP-3, and TRP-4 were expressed but only TRP-3 expression was enhanced in response to Tunicamycin. Peak response to ATP in Ca2+-free Ringers, a P2Y2 agonist for TM cells, was not affected significantly by the 6-hours treatment with tunicamycin. Subsequent exposure to 1.4 mM Ca2+-rich Ringers to elicit capacitative calcium influx (CCE), showed only transient influx (n=6). Conclusion: (1) Tunicamycin induced UPR increases transcription of CHOP while that of GRP78 and GRP94 are not affected. (2) Dexamethasone also causes ER stress presumably «ER overload response» through enhanced expression of myocilin, (3) Extent of Ca2+ in IP3-stores is not affected but a sustained increase in [Ca2+]i is not produced by CCE although TRP-3 expression is enhanced.

Keywords: 601 trabecular meshwork • 605 transcription factors • 503 outflow: trabecular meshwork 

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