Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch.
OBJECTIVE
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.
Bradley
John M B
JM
Casey Eye Institute, Oregon Health and Science University, 3375 SW Terwilliger, Portland, OR 97239-4197, USA.
Kelley
Mary J
MJ
Rose
Anastasia
A
Acott
Ted S
TS
D020121:5' Untranslated Regions / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013314:Stress, Mechanical; D013552:Swine; D014129:Trabecular Meshwork / Q000201:enzymology*
D020121:5' Untranslated Regions; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2