Transforming growth factor-β2 increases extracellular matrix proteins in optic nerve head cells via activation of the Smad signaling pathway

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.