Signaling Pathways Used in Trabecular Matrix Metalloproteinase Response to Mechanical Stretch

John M B Bradley, Mary Kelley, Anastasia Rose, Ted Acott

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

PURPOSE. Trabecular meshwork (TM) matrix metalloproteinase (MMP), and tissue inhibitor (TIMP) changes in response to mechanical stretching appear to be central to intraocular pressure (IOP) homeostasis. Studies were conducted to define the signal transduction pathway responsible for the increases in MMP-2 and -14 that occur in response to mechanical stretching of TM cells. METHODS. Porcine TM cells were subjected to mechanical stretching, and changes in MMP-2 and -14 levels were determined by gelatin zymography and Western immunoblot analysis. Effects of signal transduction pathway inhibitors on MMP levels were analyzed. Phosphospecific antibodies were used to identify phosphorylation changes in select pathway intermediates. In silico secondary structure analysis was conducted on the 5′ untranslated regions (UTRs) of MMP-2 and -14 mRNAs. RESULTS. The increases in MMP-2 and -14 that occur 24 hours after sustained mechanical stretching of TM cells were blocked by rapamycin. Wortmannin blocked the MMP-2 but not the MMP-14 increase. Protein kinase B (PKB) phosphorylation on S473 and T308 was increased significantly by stretching. Rapamycin-sensitive phosphorylation of T389 in p70/p85 S6 kinase was also increased. The phosphorylations of the translation initiation factor eIF-4E on S209 and of its inhibitory binding protein 4E-BP1 on T70 were both increased by stretch. The calculated free energies of secondary structures of the 5′ UTRs of the mRNAs for MMP-2 and -14 were negative and relatively large. MMP-2 also had pyrimidine tracts in the extreme 5′ region of its UTR. CONCLUSIONS. The increases in TM MMP-2 and -14 protein levels in response to mechanical stretching appear to be transduced at least in part by mTOR, the mammalian target of rapamycin (mTOR). The wortmannin sensitivity implicates phosphoinositide 3-kinase as a modulator of the MMP-2 but not the MMP-14 increase. Integrin-linked kinase (ILK), phosphoinositide-dependent kinase (PDK-1), and PKB are implicated in the MMP-2 increase. Translational initiation involving eIF-4E and its inhibitory binding protein 4E-BP1 appear to be involved in both the MMP-2 and -14 increases with stretching and are normally regulated by mTOR. The high degree of secondary structure in the 5′ UTRs of these transcripts is typically an indicator of genes specifically sensitive to regulation through this pathway. P70/p85 S6 kinase is probably involved downstream from mTOR and PKB in regulating translation of MMP-2, which has pyrimidine tracts in its 5′ UTR. Manipulation of these transduction pathways may provide new approaches to therapeutic IOP regulation.

Original languageEnglish (US)
Pages (from-to)5174-5181
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number12
DOIs
StatePublished - Dec 2003

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Matrix Metalloproteinase 2
Matrix Metalloproteinase 14
Matrix Metalloproteinases
Trabecular Meshwork
Sirolimus
5' Untranslated Regions
Proto-Oncogene Proteins c-akt
Phosphorylation
70-kDa Ribosomal Protein S6 Kinases
1-Phosphatidylinositol 4-Kinase
Matrix Metalloproteinase Inhibitors
Intraocular Pressure
Signal Transduction
Carrier Proteins
TOR Serine-Threonine Kinases
Phospho-Specific Antibodies
Untranslated Regions
Peptide Initiation Factors
Messenger RNA
Gelatin

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Signaling Pathways Used in Trabecular Matrix Metalloproteinase Response to Mechanical Stretch. / Bradley, John M B; Kelley, Mary; Rose, Anastasia; Acott, Ted.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 12, 12.2003, p. 5174-5181.

Research output: Contribution to journalArticle

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N2 - PURPOSE. Trabecular meshwork (TM) matrix metalloproteinase (MMP), and tissue inhibitor (TIMP) changes in response to mechanical stretching appear to be central to intraocular pressure (IOP) homeostasis. Studies were conducted to define the signal transduction pathway responsible for the increases in MMP-2 and -14 that occur in response to mechanical stretching of TM cells. METHODS. Porcine TM cells were subjected to mechanical stretching, and changes in MMP-2 and -14 levels were determined by gelatin zymography and Western immunoblot analysis. Effects of signal transduction pathway inhibitors on MMP levels were analyzed. Phosphospecific antibodies were used to identify phosphorylation changes in select pathway intermediates. In silico secondary structure analysis was conducted on the 5′ untranslated regions (UTRs) of MMP-2 and -14 mRNAs. RESULTS. The increases in MMP-2 and -14 that occur 24 hours after sustained mechanical stretching of TM cells were blocked by rapamycin. Wortmannin blocked the MMP-2 but not the MMP-14 increase. Protein kinase B (PKB) phosphorylation on S473 and T308 was increased significantly by stretching. Rapamycin-sensitive phosphorylation of T389 in p70/p85 S6 kinase was also increased. The phosphorylations of the translation initiation factor eIF-4E on S209 and of its inhibitory binding protein 4E-BP1 on T70 were both increased by stretch. The calculated free energies of secondary structures of the 5′ UTRs of the mRNAs for MMP-2 and -14 were negative and relatively large. MMP-2 also had pyrimidine tracts in the extreme 5′ region of its UTR. CONCLUSIONS. The increases in TM MMP-2 and -14 protein levels in response to mechanical stretching appear to be transduced at least in part by mTOR, the mammalian target of rapamycin (mTOR). The wortmannin sensitivity implicates phosphoinositide 3-kinase as a modulator of the MMP-2 but not the MMP-14 increase. Integrin-linked kinase (ILK), phosphoinositide-dependent kinase (PDK-1), and PKB are implicated in the MMP-2 increase. Translational initiation involving eIF-4E and its inhibitory binding protein 4E-BP1 appear to be involved in both the MMP-2 and -14 increases with stretching and are normally regulated by mTOR. The high degree of secondary structure in the 5′ UTRs of these transcripts is typically an indicator of genes specifically sensitive to regulation through this pathway. P70/p85 S6 kinase is probably involved downstream from mTOR and PKB in regulating translation of MMP-2, which has pyrimidine tracts in its 5′ UTR. Manipulation of these transduction pathways may provide new approaches to therapeutic IOP regulation.

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