Changes in gene expression by trabecular meshwork cells in response to mechanical stretching.

PURPOSE: Trabecular meshwork (TM) cells appear to sense changes in intraocular pressure (IOP) as mechanical stretching. In response, they make homeostatic corrections in the aqueous humor outflow resistance, partially by increasing extracellular matrix (ECM) turnover initiated by the matrix metalloproteinases. To understand this homeostatic adjustment process further, studies were conducted to evaluate changes in TM gene expression that occur in response to mechanical stretching. METHODS: Porcine TM cells were subjected to sustained mechanical stretching, and RNA was isolated after 12, 24, or 48 hours. Changes in gene expression were evaluated with microarrays containing approximately 8000 cDNAs. Select mRNA changes were then compared by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western immunoblots were used to determine whether some of these changes were associated with changes in protein levels. RESULTS: On the microarrays, 126 genes were significantly upregulated, and 29 genes were significantly downregulated at one or more time points, according to very conservative statistical and biological criteria. Of the genes that changed, several ECM regulatory genes, cytoskeletal-regulatory genes, signal-transduction genes, and stress-response genes were notable. These included several proteoglycans and matricellular ECM proteins composed of common repetitive binding domains. The results of analysis of mRNA changes in more than 20 selected genes by qRT-PCR supported the findings in the microarray analysis. Western immunoblots of several proteins demonstrated protein level changes associated with changes in the level of mRNA. CONCLUSIONS: The expression of a variety of TM genes is significantly affected by mechanical stretching. These include several ECM proteins that contain multiple binding sites and may serve organizational roles in the TM. Several proteins that could contribute to the homeostatic modification of aqueous humor outflow resistance are also upregulated or downregulated.

The prodomain of BMP-7 targets the BMP-7 complex to the extracellular matrix.

Biochemical and biophysical methods are used to show that BMP-7 is secreted as a stable complex consisting of the processed growth factor dimer noncovalently associated with its two prodomain propeptide chains and that the BMP-7 complex is structurally similar to the small transforming growth factor beta (TGFbeta) complex. Because the prodomain of TGFbeta interacts with latent TGFbeta-binding proteins, a family of molecules homologous to the fibrillins, the prodomain of BMP-7 was tested for binding to fibrillin-1 or to LTBP-1. The BMP-7 prodomain and BMP-7 complex, but not the separated growth factor dimer, interact with N-terminal regions of fibrillin-1. This interaction may target the BMP-7 complex to fibrillin microfibrils in the extracellular matrix. Immunolocalization of BMP-7 in tissues like the kidney capsule and skin reveals co-localization with fibrillin. However, BMP-7 immunolocalization in other tissues known to be active sites for BMP-7 signaling is not apparent, suggesting that immunolocalization of BMP-7 in certain tissues represents specific extracellular storage sites. These studies suggest that the prodomains of TGFbeta-like growth factors are important for positioning and concentrating growth factors in the extracellular matrix. In addition, they raise the possibility that prodomains of other TGFbeta-like growth factors interact with fibrillins and/or LTBPs and are also targeted to the extracellular matrix.