May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
ß1 and ß3 Integrins Use Distinct Pathways to Regulate Cross-Linked Actin Network (CLAN) Formation in Human Trabecular Meshwork (HTM) Cells
Author Affiliations & Notes
  • M. S. Filla
    University of Wisconsin, Madison, Wisconsin
    Pathology & Laboratory Medicine,
  • N. Sheibani
    University of Wisconsin, Madison, Wisconsin
    Ophthalmology & Visual Sciences,
  • P. L. Kaufman
    University of Wisconsin, Madison, Wisconsin
    Ophthalmology & Visual Sciences,
  • D. M. Peters
    University of Wisconsin, Madison, Wisconsin
    Pathology & Laboratory Medicine,
  • Footnotes
    Commercial Relationships M.S. Filla, Provisional Patent applied for, P; N. Sheibani, None; P.L. Kaufman, None; D.M. Peters, Provisional patent applied for, P.
  • Footnotes
    Support GRF (MF), EYO17006 HIGHWIRE EXLINK_ID="48:5:1145:1" VALUE="EYO17006" TYPEGUESS="GENPEPT" /HIGHWIRE , EYO16236 HIGHWIRE EXLINK_ID="48:5:1145:2" VALUE="EYO16236" TYPEGUESS="GENPEPT" /HIGHWIRE , EYO12515 HIGHWIRE EXLINK_ID="48:5:1145:3" VALUE="EYO12515" TYPEGUESS="GENPEPT" /HIGHWIRE (DP); EYO2698 (PK); EYO6665-01 (PK, DP); EY13700, RPB (NS), RRF, OPREF
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1145. doi:
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      M. S. Filla, N. Sheibani, P. L. Kaufman, D. M. Peters; ß1 and ß3 Integrins Use Distinct Pathways to Regulate Cross-Linked Actin Network (CLAN) Formation in Human Trabecular Meshwork (HTM) Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1145.

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

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Abstract

Purpose:: To identify the integrin signaling pathways regulating the cytoskeletal changes responsible for CLAN formation in HTM cells.

Methods:: FACS analysis was used to determine levels of ß1 and αvß3 integrins and integrin-associated protein (IAP) which regulates the activity of ß3 integrins. CLAN formation was induced in HTM cells by plating cells for 3hr on fibronectin-coated coverslips in the presence or absence of the ß3 integrin-activating antibody AP-5. Constituents of the integrin signaling pathways were determined by plating HTM cells in the absence or presence of the IAP-activating peptide 4N1K (100 and 200µg/mL), the IAP-blocking antibody B6H12.2 (20µg/mL), the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (20µM), the Rac1 inhibitor NSC23766 (20µM) or the Src inhibitor PP2 (5 and 20 µM). Cells were fixed and labeled with phalloidin and the percentage of CLAN-positive cells was determined using fluorescence microscopy.

Results:: HTM cells expressed similar levels of αvß3 integrins and IAP, but ß1 integrin levels were 10-fold higher than αvß3 integrin levels. Activation of either IAP or ß3 integrin by 4N1K- or AP-5 respectively caused a 2 to 3-fold increase in CLAN formation relative to untreated cells. Both 4N1K and AP-5-induced CLAN formation was blocked by the anti-IAP antibody B6H12.2 suggesting that an IAP/ß3 integrin complex may be involved. LY294002 decreased CLAN formation in cells spread on fibronectin alone (ß1 integrin-mediated) by 42% compared to control cells while CLAN formation in cells spread in the presence of antibody AP-5 (ß3 integrin-mediated) was unaffected. In contrast, ß1-mediated CLAN formation was unaffected by NSC23766 while ß3 integrin-mediated CLAN formation was reduced by 40%. CLAN formation was strongly impaired in cells treated with the Src inhibitor PP2 (5 or 20µM) regardless of whether both integrins were activated. Activation of ß3 integrins with AP-5 treatment partially rescued CLAN formation in 5µM PP2-treated cells, however no rescue was observed in cells treated with 20µM PP2.

Conclusions:: Integrin-mediated CLAN formation in HTM cells is dependent upon distinct ß1 and ß3 integrin signaling pathways. ß1-mediated CLAN formation is PI3K-dependent while ß3-mediated CLAN formation is Rac1-dependent and involves IAP. Signaling from both integrins is Src-dependent. Mapping of the signaling pathways that regulate CLAN formation may provide information regarding potential targets for new therapies to treat steroid induced glaucoma.

Keywords: trabecular meshwork • cytoskeleton • signal transduction 
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