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J. A. Fuller, A.-M. Brun-Zinkernagel, R. J. Wordinger; PACE4 Is Involved in TGF-β2 Maturation in Porcine Optic Nerve Astrocytes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3685.
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The subtilisin/kexin like proprotein convertase (SPC) family is known to process a wide variety of protein substrates. PACE4 is a secreted SPC that binds to the extracellular matrix via its cysteine rich domain (CRD). Recent research has demonstrated that furin can process TGF-β1 but does not efficiently process TGF-β2, a molecule believed to be involved in ECM remodeling of the ONH in glaucoma. The purpose of this study is to determine if modulation of PACE4 affects TGF-β2 maturation.
Primary porcine optic nerve astrocytes were subjected to oxygen glucose deprivation (OGD), an in vitro model for ischemia, for 8 hours followed by 16 hours of reperfusion. Inhibition of PACE4 expression and extracellular activity was inhibited by siRNA and treatment with 580 nM hexa-D-arginine (HDR), respectively. Immunocytochemistry was used to colocalize PACE4 and fibronectin, a component of the ECM. Analysis of PACE4 mRNA expression was performed via Quantitative Reverse Transcriptase PCR (QRT-PCR). Protein lysates were collected and immunoblotting for PACE4 was performed. SPC activity was assessed using a fluorogenic substrate following knockdown and/or activity inhibition. Cell culture supernatants from samples were used for ELISA analysis of secreted TGF-β2.
mRNA and protein expression of PACE4 increases following transient OGD. Immunocytochemistry demonstrated increased extracellular colocalization of PACE4 and fibronectin. SPC activity decreased approximately 12% following addition of 580 nM HDR. However, this decrease was abrogated upon siRNA-mediated knockdown of PACE4. OGD increases secreted active TGF-β2, whereas PACE4 siRNA and 580 nM HDR synergistically increases the ratio of latent/active TGF-β2.
This study suggests that PACE4 is upregulated following oxygen glucose deprivation. Furthermore, modulating the expression or activity of PACE4 increases the ratio of secreted latent TGF-β2. Thus, PACE4 may modulate ECM synthesis by optic nerve astrocytes, and may be a central regulator in optic nerve remodeling as observed in primary open angle glaucoma.
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