CLU is a ubiquitous glycoprotein that is especially abundant in cells at tissue-fluid interfaces and has been implicated in the maintenance of normal cell-extracellular matrix interaction.34, 35
Previous studies showed the presence of CLU in healthy human corneal endothelium.36, 37
In this study, the proteomic analysis of normal and FED HCEC-DM complexes suggests that post-translational processing and expression of CLU differ in FED tissue. Subsequently, targeted studies were performed to investigate the differential expression of specific forms of CLU in normal and FED endothelium. As a result, both the pro-survival and pro-apoptosis forms of CLU were found to be over-expressed in FED cells. This up-regulated CLU synthesis points to an undiscovered form of dysregulation of endothelial function involved in FED pathogenesis.
Several techniques were employed to characterize the differential expression of CLU forms between normal and FED specimens. When profiling the differential protein expression between normal and FED specimens, one of the most striking differences was the expression of sCLU in the 30–40 kDa range. Even though other protein differences were noted, one of them being an increased number of βIG-H3 spots in FED samples, we postponed the investigation of those differences for subsequent studies and focused on CLU. The MALDI-TOF identification of a greater number of sCLU spots in FED samples within the 30–40 kDa molecular weight range indicated that there were marked differences in the post-translational modification of sCLU in FED versus normal samples.
Western blot analysis further investigated CLU protein expression in both normal and FED endothelium. Expression of CLU in FED HCEC was significantly higher than in normal HCEC for both the nuclear and pre-secretory forms. Of interest was the finding that the level of the 30–40 kDa sCLU, which is the secreted form of CLU, was not significantly elevated in FED cells. A study by O’Sullivan et al. 28
noted that, in MCF-7 epithelial cells, the proteolytic cleavage required to produce the mature secretory form of CLU occurs in the Golgi prior to extracellular secretion. Stressing the cells with pro-apoptotic stimuli, such as TNF-α, blocked the proteolysis of pre-sCLU in the Golgi and prevented the formation of the secreted α and β chains. Therefore, it is possible, that FED cells have an alteration in the post-translational modification of pre-sCLU, preventing a parallel increase in pre-sCLU and sCLU. Similarly, Nizard et al. 38
showed that, under certain stressed conditions, CLU can evade the secretion pathway altogether and localize mainly within the cytosol, where it exerts its biological functions. Separate studies have shown that the intracellular 60 kDa form of CLU, and not the secretory 40 kDa isoform, is responsible for the anti-apoptotic effects of CLU by interfering with Bax activation in mitochondria.39
FED-affected endothelial cells were also found to have elevated levels of nCLU as compared to the normal cells. The nuclear CLU 49 kDa band was consistently present in FED, but not in normal cells by Western blotting. These elevated levels of nCLU correlated with increased nuclear staining of CLU in FED-affected cells by confocal microscopy, indicating that increased production of nCLU is followed by its translocation to the nucleus in the pathological state, but not in the normal cells. These data are in agreement with previous studies that showed induction and translocation of CLU from the cytoplasm to the nucleus after cytotoxic stimulation with IR and TGF-β treatment.25, 27
Separate studies have shown that over-expression of nCLU without cytotoxic stimulation leads to cell death, pointing out its role in apoptosis independent of exogenous causes.15, 40
In the nucleus, nCLU has been shown to interact with the Ku70 subunit of the Ku70/80 protein, which is involved in DNA double-strand break repair.15, 17, 27
When bound to the over-expressed nCLU, Ku70/80 is prevented from DNA end-binding, thus preventing repair of genomic breaks and leading to genomic instability. Although additional information is needed regarding how nCLU affects the DNA repair process, it is known, that over-expression of nCLU causes diminished cell growth and leads to lethality.15
To investigate whether the mRNA level of CLU increases in FED cells, RT-PCR analysis was performed. We used a well-established primer set, which amplifies all four CLU exons and detects the full length form of CLU.27
There was a two-fold increase in CLU cDNA in FED cells vs. normals, indicating that there is an overall increase in CLU mRNA, as well as protein, expression in the diseased cells. The RT-PCR and Western blot data was identical regardless of storage time and the use of single or pooled samples. In additional experiments (data not shown), we used a primer set reported to specifically amplify the nuclear form of CLU27
, but could not obtain consistent results—a finding similar to that of other investigators (personal communication with Dr. Denis Michel, Université de Rennes, Rennes, France).38
The mRNA analysis of the tissue taken from the pseudopakic bullous keratopathy specimens revealed a relative decrease in CLU production as compared to normal specimens, indicating that a similar increase in CLU mRNA expression does not occur in PBK. Such findings indicate that CLU overexpression in FED may be specific to the pathogenesis of the dystrophy and not seen under other corneal swelling conditions.
Confocal microscopy revealed an unusual CLU staining pattern in the FED endothelium. CLU exhibited mostly intracellular staining, which was highlighted at the edges of the cell membranes next to dark circular areas suggestive of guttae. The central fluorescence within those dark areas could represent CLU expression in the remnants of dying cells. It is also possible that the unusual staining pattern in those areas signifies the propensity of CLU to associate with dead cells lacking intact cell membranes, as shown to occur in L929-pRc.clus cells in response to TNF-α stimulation.41
The physiological relevance of such an interaction is not known.
In FED tissue endothelial cell nuclei clustered densely around the guttae, and those cells had an enhanced CLU staining at the cell membrane borders next to guttae. Such a staining pattern most likely represents CLU’s essential role in eliciting endothelial cell clustering under stressed conditions. Previous studies showed that CLU induced cell aggregation in response to oxidant injury due to hydrogen peroxide. The resultant CLU-induced cell aggregation was shown to protect the cells against injury by decreasing the amount of cell membrane accessible to oxidant injury and by maintaining better cell to cell contacts, that, when disrupted, can lead to apoptosis.42, 43
The finding that CLU is over-expressed in FED appears to be important in elucidating its pathophysiology. Over-expression of pre-sCLU may be a stress-induced response to protect the cells from apoptosis. Numerous studies have shown that levels of CLU are often elevated in response to a variety of tissue insults. 24, 44, 45
The prevailing thought is that CLU can act as an intracellular and extracellular chaperone and protect a variety of proteins from stress-induced precipitation by affecting their folding state.18,19
CLU has been shown to play a role in protection of kidney from ischemic glomerular injury 46, 47
; and cancer cells from apoptosis induced by chemotherapeutic agents.39,45
CLU is also over-expressed in many pathologic conditions, two of which are Alzheimer’s disease (AD) and age-related macular degeneration (ARMD).21, 48
Similar to FED, both of these disorders manifest with high amounts of extracellular membrane deposits (i.e. drusen and amyloid plaques) and concomitant dysfunction and apoptosis of the cells next to the deposits. Initial studies of AD showed that CLU protects neurons from amyloid plaque formation in vitro 47, 49
, however, in a mouse model of AD, CLU promoted amyloid plaque accumulation and neuron toxicity.50
Similarly, in the ARMD model, large amounts of CLU found in drusen were thought to promote the formation of these β-amyloid-like deposits.51
Although the exact function of CLU is not clear, the findings of CLU dysregulation in numerous pathological states point to a potentially common downstream pathway in these processes. Most studies do arrive at the same consensus though, and that is that CLU’s chaperone-like properties may induce alterations in the equilibrium between the deposited and cleared material.50
The relationship between the levels of sCLU and nCLU is not completely understood, especially how it can promote and inhibit cell death depending on the isoform expression. In colorectal carcinoma, there is a diminished expression of nCLU and increasing expression of sCLU with increasing cancer grade.20
Other studies have shown that pro-apoptotic stimuli, like IR, increase the levels of both nCLU and pre- and sCLU proteins, the latter two forms showing a much higher increase than the former.15, 52
In the IR model, pre-sCLU and sCLU levels increased with low, nontoxic, growth stimulatory levels of IR, and nCLU levels increased with much higher levels of the cytotoxic stress.15
Similarly to the IR-induced CLU overexpression, both pre-secretory and nuclear isoforms were elevated in FED. Although the driving force for CLU production in FED is yet to be elucidated, one of the potential factors might be oxidative stress. Numerous studies have shown that oxidative stress and reactive oxygen species can induce CLU overproduction and that CLU can render the cells resistant to reactive oxygen species-mediated cellular injury.24, 42, 53
There is mounting evidence in the current literature that oxidative stress plays a role in FED.54, 55
Therefore, it is possible that dysregulation of CLU production is indirectly pointing to the mechanism of FED pathogenesis involving oxidative stress-induced damage to the corneal endothelium.