The transforming growth factor beta induced protein is mainly an extracellular matrix protein that has a secretory signal sequence at the N-terminus, four homologous internal domains and a cell attachment site (consisting of RGD amino acids) at the C-terminus.6
The main function of TGFBIp is attributed to its ability to bind various ligands and mediate cell adhesion via the RGD sequence.7
Our previous studies detected a marked overexpression of clusterin and TGFBIp in FECD endothelium.3
In the current study, we performed a targeted analysis of TGFBIp processing in normal and FECD HCEC-DM and compared TGFBIp expression in young and old donors. The co-localization of TGFBIp and CLU expression in guttae provided us with an insight into the structural composition of these extracellular excrescences.
Proteomic analysis of normal HCEC-DM showed that TGFBIp is abundantly expressed in the endothelial-DM complex. TGFBIp has multiple threonine, tyrosine and serine sites which allow phosphorylation and thus posttranslational modification.16
On 2-D gels, 68- and 39-kDa TGFBIp fragments migrated as series of spots with different pI’s most likely representing different postranslational modifications of TGFBIp. This finding is in agreement with the other studies that performed 2-D electrophoresis on the whole corneal extracts and identified TGFBIp in multiple spots migrating at the same molecular weight, but with different pI’s.16
The fact that the 57- and 29-kDa fragments were repeatedly identified by Western blotting but not on the 2-D gels is most likely due to these forms being present in much lower concentrations and to MALDI-TOF not being sensitive enough to identify them.
The analysis of TGFBIp content using antibodies against two non-overlapping parts of the protein revealed the presence of C-terminal fragments of the protein that varied in relative concentration with increasing age of the donors. In addition to the major 68-kDa band corresponding to the full-size protein, we identified a set of fragments that are most likely the proteolysis products characteristic for TGFBIp turnover in normal HCEC-DM. It is possible that the TGFBIp fragments were created from degradation by extracellular proteases. Nevertheless, a more plausible explanation is the fragment formation from normal TGFBIp turnover in corneal endothelium, since almost identical molecular weight fragments were detected in the study by Korvatksa et al., in which N-terminal sequencing and immunostaining were performed to characterize TGFBIp content in the whole corneas.16
Our study showed an increase in the levels of the 39-kDa fragment with increasing age of donors. Of note, only 68-and 39-kDa bands were present throughout the age range of donors. The other fragments (57-kDa and 29-kDa) appeared to be present in older age samples only, implicating the age-related differences in normal TGFBIp turnover. The gradual thickening of Descemet’s membrane and building of the posterior nonbanded layer with age is in agreement with increasing accumulation of TGFBIp which is known to have a physiological interaction with collagens.7, 17, 18
Previous studies performed on whole rabbit corneal buttons showed an increase in steady state levels of TGFBI
mRNA that correlated with increasing rate of collagen accumulation during corneal morphogenesis.18
Several immunohistochemical studies have localized TGFBIp in normal Descemet’s membrane, posterior collagenous layer, and retrocorneal fibrous membrane inferring its role as a structural element of the aging and injured Descemet’s membrane.19–22
The exact role of TGFBIp is not known but its abundance in the extracellular milieu of endothelium implies a substantial role in the cell-Descemet’s membrane interaction.
Previous study comparing 2-D gels of FECD and normal donors revealed an increase in TGFBIp spot intensity and number in FECD samples.3
To analyze this difference further we performed Western blot analysis comparing the TGFBIP expression in normal and FECD HCEC-DM. Expression of 68-, 57-, and 39-kDa fragments was statistically significantly higher in the FECD HCEC-DM. The expression of the 29-kDa fragment was elevated in FECD but not at a significant level. To corroborate the increase of TGFBIp levels in FECD, its expression at the gene level was compared between normal and FECD samples. The finding that levels of TGFBI
mRNA were significantly increased in FECD samples further substantiates the proteomic data and indicates that the source of the differences stem from increased gene transcription.
Studies analyzing pathologic stromal deposits due to mutation in TGFBI
gene have also identified an accumulation of TGFBIp fragments in dystrophic corneas as compared to normal controls.16, 23, 24
TGFBIp has been shown to co-localize and co-aggregate into these deposits which were composed of either amyloid or non-amyloid depending on the form of stromal dystrophy.16
In addition to the increase in TGFBIp production, the analysis of these corneas detected overexpression of aberrant forms of TGFBIp that varied in their molecular weight depending on the type of dystrophy. In our study of FECD samples, we did not detect any aberrant or unusual forms of TGFBIp that were not present in normal age-matched controls, implying that probably there is no intrinsic mutation in the TGFBI
gene causing the accumulation of the protein. This is in contrast to the findings in the stromal dystrophies where overexpression of both normal and aberrant forms of TGFBIp has been attributed to the TGFBI
The immunohistological analysis revealed an interesting pattern of CLU and TGFBIp colocalization in the guttae, the excrescences characteristic of Fuchs corneal dystrophy. The staining for TGFBIp was prominent throughout the Descemet’s membrane and showed a marked increase in intensity at the centers of the guttae. CLU was also present in the centers of the guttae but on top of TGFBIp staining, closer to the apical side of the endothelium. The diagram in shows a schematic representation of the relationship of CLU and TGFBIp role in guttae formation. This is the first known study co-localizing CLU and TGFBIp in these pathologic extracellular matrix deposits. There is no known correlation in the literature between clusterin and TGFBIp. It is known though, that CLU overexpression at the times of cellular stress (including oxidative stress) causes the cells to aggregate via cell-cell and cell-substratum interactions.5
The driving force of these interactions often times is the production of cell adhesive molecules and junctional complexes. In a renal injury model, such interactions are capable of preserving the integrity of the renal epithelial barrier.25, 26
When the cell-matrix interactions are disrupted, a form of apoptosis called ‘anoikis’ ensues. Similarly in FECD, there is an over-production of the cell adhesion molecule, TGFBIp in the setting of the prolonged tissue injury and pathological CLU overexpression. It is possible, that the endothelial cells under stressors of the dystrophic degeneration are ‘clumping’ with one another via the action of CLU and are adhering to their substratum via the action of TGFBIp. The attempt of the cells to attach to their substratum is progressively disrupted during guttae formation, thus rendering the cells susceptible to apoptosis.
Schematic representation of the role TGFBIp and CLU in guttae formation
In conclusion, there is an increased production and modification of TGFBIp in the aging HCEC-DM complex. The increase in TGFBIp production is even greater in FECD as compared to the age-matched normal controls. The pro-aggregative protein CLU, and pro-adhesive protein TGFBIp, co-localize in the centers of guttae. Even though such findings cannot infer the functional role of these proteins in the pathogenesis of the dystrophy, it provides us with the better understanding of the major culprits involved in the aberrant cell-extracellular matrix interactions.