TY - JOUR
T1 - TGFβ2 induces the formation of cross-linked actin networks (CLANs) in human trabecular meshwork cells through the smad and non-smad dependent pathways
AU - Montecchi-Palmer, Michela
AU - Bermudez, Jaclyn Y.
AU - Webber, Hannah C.
AU - Patel, Gaurang C.
AU - Clark, Abbot F.
AU - Mao, Weiming
N1 - Funding Information:
Supported by National Eye Institute Grant R21EY023048 (W.M.).
Publisher Copyright:
© 2017 The Authors.
PY - 2017/2
Y1 - 2017/2
N2 - PURPOSE. Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation. METHODS. Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFb receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4’,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett’s post hoc test. RESULTS. TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFb receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. CONCLUSIONS. TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.
AB - PURPOSE. Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation. METHODS. Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFb receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4’,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett’s post hoc test. RESULTS. TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFb receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. CONCLUSIONS. TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.
KW - Cross-linked actin networks
KW - Glaucoma
KW - TGFβ2
KW - Trabecular meshwork
UR - http://www.scopus.com/inward/record.url?scp=85014075384&partnerID=8YFLogxK
U2 - 10.1167/iovs.16-19672
DO - 10.1167/iovs.16-19672
M3 - Article
C2 - 28241317
AN - SCOPUS:85014075384
VL - 58
SP - 1288
EP - 1295
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 2
ER -