TGFβ2 induces the formation of cross-linked actin networks (CLANs) in human trabecular meshwork cells through the smad and non-smad dependent pathways

Michela Montecchi-Palmer, Jaclyn Y. Bermudez, Hannah C. Webber, Gaurang C. Patel, Abbot Clark, Weiming Mao

Research output: Contribution to journalArticleResearchpeer-review

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Abstract

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.

Original languageEnglish
Pages (from-to)1288-1295
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume58
Issue number2
DOIs
StatePublished - 1 Feb 2017

Fingerprint

Trabecular Meshwork
Transforming Growth Factors
Actins
Extracellular Signal-Regulated MAP Kinases
JNK Mitogen-Activated Protein Kinases
Stress Fibers
Aqueous Humor
Phalloidine
rho-Associated Kinases
Intraocular Pressure
Protein Kinases
Analysis of Variance

Keywords

  • Cross-linked actin networks
  • Glaucoma
  • TGFβ2
  • Trabecular meshwork

Cite this

Montecchi-Palmer, Michela ; Bermudez, Jaclyn Y. ; Webber, Hannah C. ; Patel, Gaurang C. ; Clark, Abbot ; Mao, Weiming. / TGFβ2 induces the formation of cross-linked actin networks (CLANs) in human trabecular meshwork cells through the smad and non-smad dependent pathways. In: Investigative Ophthalmology and Visual Science. 2017 ; Vol. 58, No. 2. pp. 1288-1295.
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abstract = "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.",
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TGFβ2 induces the formation of cross-linked actin networks (CLANs) in human trabecular meshwork cells through the smad and non-smad dependent pathways. / Montecchi-Palmer, Michela; Bermudez, Jaclyn Y.; Webber, Hannah C.; Patel, Gaurang C.; Clark, Abbot; Mao, Weiming.

In: Investigative Ophthalmology and Visual Science, Vol. 58, No. 2, 01.02.2017, p. 1288-1295.

Research output: Contribution to journalArticleResearchpeer-review

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

AU - Mao, Weiming

PY - 2017/2/1

Y1 - 2017/2/1

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

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JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

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