Transforming growth factor-β2 induces synthesis and secretion of endothelin-1 in human trabecular meshwork cells.
OBJECTIVE
Analysis of aqueous humor from patients with primary open-angle glaucoma (POAG) revealed marked increases in the content of endothelin-1 (ET-1) and transforming growth factor-beta (TGF-β). We determined the consequences of TGF-β signaling on ET-1 expression and secretion by human trabecular meshwork (TM) cells.


METHODS
Primary or transformed (NTM5 and GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-β1 or -β2. Relative changes in preproendothelin (ppET)-1 mRNA content and secreted ET-1 peptide were quantified by real-time PCR and ELISA, respectively. In some experiments, TGF-β or ET-1 receptor antagonists, or Rho G-protein inhibitors, were evaluated for effects on TGF-β signaling. Filamentous actin organization was visualized by phalloidin.


RESULTS
Primary or transformed human TM cells cultured in the presence of TGF-β1 or -β2 exhibit a marked (>8-fold) increase in ppET-1 mRNA content compared to vehicle controls. Coincubation with SB-505124, an inhibitor of TGFβRI/ALK-5 signaling, prevented TGF-β-mediated ppET-1 mRNA expression. In contrast, coincubation with ET(A) (BQ-123) or ET(B) (BQ-788) receptor antagonists had no effect on TGF-β-mediated ppET-1 mRNA expression. TGF-β1 and -β2 each elicited a robust (>7-fold) secretion of ET-1 while enhancing stress fiber organization. Inhibition of Rho signaling attenuated TGF-β-mediated increases in ppET-1 mRNA content, ET-1 secretion, and stress fiber organization.


CONCLUSIONS
TGF-β, signaling through the TGFβRI/ALK-5 receptor, elicits marked increases in ET-1 mRNA content and ET-1 secretion from cultured primary or transformed human TM cells. Elevated levels of TGF-β2 present in AH of POAG patients may elevate intraocular pressure, in part, by eliciting aberrant Rho G-protein dependent cell contraction, and increasing ET-1 synthesis and secretion, in human TM cells.
Research Service, Department of Veterans Affairs, Edward Hines Jr. VA Hospital, Hines, Illinois, USA.
Cynthia L
CL
Von Zee
Kelly A
KA
Langert
Evan B
EB
Stubbs
D000199:Actins / Q000378:metabolism; D000704:Analysis of Variance; D052117:Benzodioxoles / Q000494:pharmacology; D002102:Cadaver; D002461:Cell Line, Transformed; D019332:Endothelin-1 / Q000096:biosynthesis* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008148:Lovastatin / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins; D015398:Signal Transduction; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D053781:Transforming Growth Factor beta2 / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*
C519132:2-(5-benzo(1,3)dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride; D000199:Actins; D052117:Benzodioxoles; D019332:Endothelin-1; D007093:Imidazoles; D011725:Pyridines; D012333:RNA, Messenger; D011994:Recombinant Proteins; D053781:Transforming Growth Factor beta2; D008148:Lovastatin