MMP and TIMP Expression in Quiescent, Dividing, and Differentiating Human Lens Cells

Abstract

PURPOSE.Matrix metalloproteinases (MMPs) and the tissue inhibitors of the MMPs (TIMPs) have been implicated in lens differentiation, growth, remodeling, and cataract. Hence, a gene expression analysis was undertaken in epithelial and fiber cells dissected from clear human donor lenses. METHODS. The human lens was dissected into three regions: anterior epithelial, equatorial, and fiber cells. Primary lens cell cultures were also analyzed. cDNA was generated by reverse transcription of the mRNA portion of the total RNA isolated from each sample. Gene expression data were generated using quantitative real-time reverse transcription PCR. Data were analyzed in terms of cycle threshold number (C T ) and were normalized to endogenous 18S expression. Western blot analyses were carried out to confirm the presence of two critical MMPs. RESULTS. Anterior and equatorial samples were uncontaminated by fiber cells because they showed high expression of ␣-crystallin genes but low expression of ␤-and ␥-crystallins. The fibers had high expression of these genes and of MIP. MMP genes were expressed at uniformly low levels in the native tissues except for MMP-14 and -15 (MT1-and MT2-MMP, respectively). In fact, MT1-MMP declined in expression from the anterior epithelium to fibers, whereas MT2-MMP increased. The presence of MT1 and MT2-MMP proforms and faster migrating bands, indicating processed or activated forms, was confirmed at the protein level. TIMP genes were uniformly highly expressed in native tissues, with TIMP-3 having the highest expression in the epithelial tissues and TIMP-2 in the fibers. MMP expression was generally elevated in both sets of cultured cells, including MMP-2 and -9. TIMP genes were also relatively highly expressed in the cultured cells. CONCLUSIONS. MMP expression is generally well regulated in native tissues, with relatively low expression of MMPs and high expression of TIMPs. Membrane-type MMPs (MT1 and 2-MMPs) were the most highly expressed; this is important in a tissue with relatively high membrane content but low extracellular space. The striking reciprocal patterns of expression of MT1-MMP and MT2-MMP indicate that these enzymes are of particular significance in lens function. (Invest Ophthalmol Vis Sci.

MMP2 Activity is Critical for TGFβ2-Induced Matrix Contraction—Implications for Fibrosis

“…The role of MT6-MMP remains to be determined, but MT2-MMP is expressed in human anterior lens epithelium along with MT1-MMP, MT3-MMP, MT4-MMP, and MT5-MMP. 24 A study by Morrison et al 46 using TIMP2 knockout mice fibroblast cells revealed that heightened levels of MT2-MMP were sufficient to activate pro-form MMP2 despite the lack of TIMP2. This was dependent on MMP2 binding to the cell surface via the hemopexin domain most likely by association with integrin avb6.…”

“…23 This study also demonstrated that the broadspectrum MMP inhibitor GM6001 and a specific MMP2/9 inhibitor reduced the formation of TGFb-induced ASC. 23 MT1-MMP (MMP14) expression is also reported in the lens 24 and is generally proposed to play a role in the activation of MMP2 through proteolytic cleavage to remove the pro-domain. 25 MT1-MMP, pro-form MMP2, and tissue inhibitor of matrix metalloproteases 2 (TIMP2) form a trimolecular complex, 26 which results in removal of the inhibitory pro-domain from MMP2 by an adjacent free MT1-MMP molecule.…”

“…Following serum starvation, cells were treated for a further 24 hours with nonsupplemented SF EMEM (control) or TGFb2 (10 ng/mL). Total RNA was subsequently prepared using an RNeasy minikit (Qiagen, West Sussex, UK) and quantitative real-time PCR was used to analyze mRNA expression of MMP family members as described by Hodgkinson et al 24 ; for MMP and TIMP primer/probe sets used see Nuttall et al 30 ELISA Analysis of Total MMP2 Protein Levels FHL-124 cells were seeded and maintained as described for oligonucleotide microarray analysis. Following serum starvation cells were placed into experimental conditions for a further 48 hours.…”

Type I Collagen Accelerates the Spreading of Lens Epithelial Cells through the Expression and Activation of Matrix Metalloproteinases

“…20,51 Unlike other MMPs, MMP-2 is constitutively expressed by many cell types. 52 Although expression of MMP2 is low within the native human lens and requires disruption to invoke upregulation, 53 our results showed that chicken LECs expressed MMP-2 in the absence of stimulation. The high levels of MMP-2 expression seen in many tissues suggest that the regulation of proenzyme activation is more relevant for the control and induction of MMP-2 action than that of other members of the MMP family in which transcriptional control is more important.…”

“…58 MT2-MMP is expressed in the human lens and primary lens cell cultures. 53 These results suggest that different mechanisms may involve MMP-2 activation in LECs.…”