TY - JOUR
T1 - Transforming growth factor-β2 increases extracellular matrix proteins in optic nerve head cells via activation of the smad signaling pathway
AU - Zode, Gulab S.
AU - Sethi, Anirudh
AU - Brun-Zinkernagel, Anne Marie
AU - Chang, I. Fen
AU - Clark, Abbot F.
AU - Wordinger, Robert J.
PY - 2011
Y1 - 2011
N2 - Purpose: Transforming growth factor-β2 (TGF-β2) is associated with glaucomatous neuropathy, primarily via the increased synthesis and secretion of extracellular matrix (ECM) proteins and remodeling of the optic nerve head (ONH). Here, we investigated the signaling pathways used by TGF-β2 to stimulate ECM expression by ONH astrocytes and lamina cribrosa (LC) cells. Methods: TGF-β2 localization and secretion was examined in human donor tissues and ONH astrocytes and LC cells. To examine TGF-β2 signaling, ONH astrocytes and LC cells were treated with recombinant TGF-β2, and phosphorylation of Smad and non-Smad signaling proteins were examined by western blot analyses and immunostaining. Results: TGF-β2 is significantly increased in the LC region of the ONH in glaucomatous eyes compared to age-matched normal eyes (n=4, p<0.0013). ONH astrocytes and LC cells secrete TGF-β2, indicating that these cells may be an in vivo source of TGF-β2 in the human ONH. In addition, treatment of ONH astrocytes and LC cells with exogenous TGF-β2 increased ECM protein synthesis and secretion. With respect to TGF-β2 signaling, recombinant TGF-β2 induced phosphorylation of canonical signaling proteins Smad2/3 but did not alter phosphorylation of non-canonical signaling proteins extracellular signal-regulated kinases (ERK)1/2, p38, and c-Jun N-terminal kinases (JNK)1/2 proteins in ONH astrocytes and LC cells. Exogenous TGF-β2 increased co-localization of pSmad2/3 with Co-Smad4 in the nucleus of ONH astrocytes and LC cells further indicating activation of the canonical Smad signaling pathway. Furthermore, inhibition of TGF-β I receptor activity by SB431542 or inhibition of Smad3 phosphorylation by SIS3 blocked TGF-β2 stimulated ECM expression as well as activation of downstream canonical pathway signaling molecules. Knockdown of either Smad2 or Smad3 via small interfering RNA (siRNA) reduced TGF-β2 stimulated ECM proteins in ONH astrocytes and LC cells. Conclusions: These studies indicate that TGF-β2 utilizes the canonical Smad signaling pathway to stimulate ECM synthesis in human ONH cells. Our studies also indicate that pSmad2/3 is required for TGF-β2 stimulation of ECM remodeling.
AB - Purpose: Transforming growth factor-β2 (TGF-β2) is associated with glaucomatous neuropathy, primarily via the increased synthesis and secretion of extracellular matrix (ECM) proteins and remodeling of the optic nerve head (ONH). Here, we investigated the signaling pathways used by TGF-β2 to stimulate ECM expression by ONH astrocytes and lamina cribrosa (LC) cells. Methods: TGF-β2 localization and secretion was examined in human donor tissues and ONH astrocytes and LC cells. To examine TGF-β2 signaling, ONH astrocytes and LC cells were treated with recombinant TGF-β2, and phosphorylation of Smad and non-Smad signaling proteins were examined by western blot analyses and immunostaining. Results: TGF-β2 is significantly increased in the LC region of the ONH in glaucomatous eyes compared to age-matched normal eyes (n=4, p<0.0013). ONH astrocytes and LC cells secrete TGF-β2, indicating that these cells may be an in vivo source of TGF-β2 in the human ONH. In addition, treatment of ONH astrocytes and LC cells with exogenous TGF-β2 increased ECM protein synthesis and secretion. With respect to TGF-β2 signaling, recombinant TGF-β2 induced phosphorylation of canonical signaling proteins Smad2/3 but did not alter phosphorylation of non-canonical signaling proteins extracellular signal-regulated kinases (ERK)1/2, p38, and c-Jun N-terminal kinases (JNK)1/2 proteins in ONH astrocytes and LC cells. Exogenous TGF-β2 increased co-localization of pSmad2/3 with Co-Smad4 in the nucleus of ONH astrocytes and LC cells further indicating activation of the canonical Smad signaling pathway. Furthermore, inhibition of TGF-β I receptor activity by SB431542 or inhibition of Smad3 phosphorylation by SIS3 blocked TGF-β2 stimulated ECM expression as well as activation of downstream canonical pathway signaling molecules. Knockdown of either Smad2 or Smad3 via small interfering RNA (siRNA) reduced TGF-β2 stimulated ECM proteins in ONH astrocytes and LC cells. Conclusions: These studies indicate that TGF-β2 utilizes the canonical Smad signaling pathway to stimulate ECM synthesis in human ONH cells. Our studies also indicate that pSmad2/3 is required for TGF-β2 stimulation of ECM remodeling.
UR - http://www.scopus.com/inward/record.url?scp=79959971614&partnerID=8YFLogxK
M3 - Article
C2 - 21738403
AN - SCOPUS:79959971614
SN - 1090-0535
VL - 17
SP - 1745
EP - 1758
JO - Molecular Vision
JF - Molecular Vision
ER -