Lysosomal dysfunction of corneal fibroblasts underlies the pathogenesis of Granular Corneal Dystrophy Type 2 and can be rescued by TFEB

Abstract Granular corneal dystrophy type 2 (GCD2) is the most common form of transforming growth factor β‐induced (TGFBI) gene‐linked corneal dystrophy and is pathologically characterized by the corneal deposition of mutant‐TGFBIp. The defective autophagic degradation of pathogenic mutant‐TGFBIp has been shown in GCD2; however, its exact mechanisms are unknown. To address this, we investigated lysosomal functions using corneal fibroblasts. Levels of cathepsins K and L (CTSK and CTSL) were significantly decreased in GCD2 cells, but of cathepsins B and D (CTSB and CTSD) did not change. The maturation of the pro‐enzymes to their active forms (CTSB, CTSK and CTSL) was inhibited in GCD2 cells. CTSL enzymes directly degraded both LC3 (autophagosomes marker) and mutant‐TGFBIp. Exogenous CTSL expression dramatically reduced mutant‐TGFBIp in GCD2 cells, but not TGFBIp in WT cells. An increased lysosomal pH and clustered lysosomal perinuclear position were found in GCD2 cells. Transcription factor EB (TFEB) levels were significantly reduced in GCD2 cells, compared to WT. Notably, exogenous TFEB expression improved mutant‐TGFBIp clearance and lysosomal abnormalities in GCD2 cells. Taken together, lysosomal dysfunction in the corneal fibroblasts underlies the pathogenesis of GCD2, and TFEB has a therapeutic potential in the treatment of GCD2.

epithelium, a thick stroma with keratocytes also known as corneal fibroblasts and the inner endothelium. 3 Keratocytes are normally quiescent and generally only become activated after corneal injury. 3 The role of keratocytes in the corneal stroma is to maintain corneal transparency and structures through the degradation and synthesis of cornea-specific extracellular matrix (ECM) components 4 ;damage to these cells may result in impaired vision.
Autophagy is a major intracellular degradation and recycling system that is ubiquitous in eukaryotes. This catabolic process is a response to cellular stress and pathophysiological conditions, whereby cellular organelles and components are engulfed into double-membrane vesicles called autophagosomes and eventually delivered to lysosomes for degradation. 4,5 The final step of autophagy is the degradation of the cargo molecules within the lysosomes.
Lysosomes are membrane-enclosed cellular organelles that consist of two types of lysosomal proteins: lysosomal acid hydrolases and lysosomal membrane proteins. The lysosomal acid hydrolases are involved in the degradation of lysosome cargo and there are more than 60 different types that degrade proteins, nucleic acids, carbohydrates and lipids. 6 Among them, the cathepsins (CTSs) are a major class of lysosomal protease, which are especially important for autophagy. [7][8][9] CTSs family consists of aspartic, cysteine and serine CTSs, and they are synthesized as immature (inactive) pro-CTSs that are proteolytically processed to form mature (active) CTSs. 10,11 Most lysosomal CTSs are functionally processed to their mature forms (or active form) at acidic pH, as CTSs are stable and active at a low pH.
TGFBIp, which is ubiquitously expressed, 12 enters through the autophagy pathway 13 and caveolae-mediated endocytosis 14 into lysosomes, where the proteins are degraded. In GCD2 corneal fibroblasts, mutant-TGFBIp accumulates in the lysosomal compartments due to defective autophagy. 13,[15][16][17] Furthermore, this accumulation of mutant-TGFBIp leads to cell death of corneal fibroblasts. 13 Several studies have found that the reduction and clearance of mutant-TGFBIp in the corneal fibroblasts is a viable therapeutic strategy for the treatment of TGFBI-linked dystrophic patients. [16][17][18][19] However, there are no therapies or drugs currently available for the treatment of GCD2.
We hypothesized that the accumulation of the pathogenic mutant-TGFBIp in the corneal fibroblasts may be caused by lysosomal abnormalities and that the enhancement of lysosomal function might counteract the progression of TGFBI-linked corneal dystrophies. In this study, we tested the lysosomal function of the corneal fibroblasts and the therapeutic effects of exogenous TFEB expression and evaluated the efficacy of this treatment strategy on GCD2 corneal fibroblasts.

| Antibodies, inhibitors and treatments
All antibodies, reagents and inhibitors that were used in this investigation are listed in Tables S1 and S2. All inhibitors and drugs were dissolved in dimethyl sulfoxide. After 16-24 hours subcultures, corneal fibroblasts were treated with each inhibitor and the relevant drugs in the fresh growth medium.

| Culture of corneal fibroblasts
Primary cultured corneal fibroblasts were prepared from heterozygous (HT) or homozygous (HO) GCD2 patients and normal (wild-type, WT) healthy corneas from the eye bank, which were obtained during penetrating or lamellar keratoplasty. Donor confidentiality was maintained according to the Declaration of Helsinki and was approved by Severance Hospital IRB Committee (CR04124), Yonsei University. GCD2 was diagnosed by DNA sequencing analysis of BIGH3 gene mutations. This study used WT (n = 4), HT (n = 1), and HO (n = 3) human corneal fibroblasts, which were immortalized by expression of the catalytic subunit of human telomerase (hTERT). 14

| CTSL and TFEB retrovirus plasmid construction and transduction
Each human full-length TFEB ORF cDNA clone was obtained from OriGene Technologies (SC122773: OriGen Rockville, MD, USA) and the human full-length CTSL ORF cDNA cloned from the total RNA of the corneal fibroblasts were cloned into the pcDNA3.1 TOPO vector (Invitrogen, Carlsbad, CA, USA) using standard RT-PCR technology. Supporting information provides more additional detail methods (Methods S1).
For immunoprecipitation, cell lysates were each divided into two equal concentrations and immunoprecipitated with anti-TFEB or anti-14-3-3 and Dynabeads coated with sheep anti-mouse IgG (Invitrogen).
The immunoprecipitated proteins were analysed by Western blots.

| Immunofluorescence staining and confocal microscopy
Corneal fibroblasts were grown on culture slides (BD Falcon Labware, REF 354108) that were permeabilized and fixed in methanol at −20°C for 3 minutes. Supporting Material provides more detail methods (Methods S3).

| Acridine orange staining
It has been established that acridine orange accumulates in acidic organelles. 20 Cells were cultured on a cover glass slide chamber, followed by the designated treatments. Briefly, corneal fibroblasts were exposed to 0.5 μg/mL acridine orange (Sigma-Aldrich) for 15 minutes at 37°C. 21 After washing with PBS three times to remove excess acridine orange, the lysosomal structures were visualized with a Zeiss LSM700 confocal microscope (Carl Zeiss).

| Determination of the lysosomal pH with a pH indicator
Changes in lysosomal pH were evaluated using the LysoSensor Yellow/Blue DND-160 reagents following the manufacturer's instructions. Cells (1 × 10 6-7 /mL) were exposed to Lysosensor yellow/blue DND-160 (Invitrogen) at a final concentration of 10 μmol/L for 1 hour in PBS. Cells were then washed three times with ice-cold PBS and kept on ice until just before starting observation.

| RNA isolation and quantitative real-time PCR
Total RNA was isolated as described by the manufacturer using Trizol reagent (Invitrogen). Quantitative real-time PCR (RT-qPCR) was performed using Taq-Man ® Universal PCR Master Mix II (Applied Biosystems) with specific primers (Table S3). The mRNA levels β-actin and GAPDH were used to normalize the expression of target genes. Relative quantification was performed using system software based on the 2 −DDCt method.

| Subcellular fractions
Nuclear fractions and the cytoplasm were prepared using a NE-PER® nuclear and cytoplasmic extraction reagents (Pierce, 78833) according to the manufacturer's protocol. Supporting information provides more detail methods (Methods S4).

| In vitro cleavage assays of TGFBIp and LC3
Human CTSL (C6854: Sigma-Aldrich) was used to digest TGFBIp and LC3 proteins in vitro. Supporting information provides more detail methods of in vitro cleavage assay (Methods S5).

| Imaging
For analysis of the TFEB in the nucleus, Z-stack images were captured at ×20 and ×60 magnifications using the Zeiss LSM 700 confocal microscope (Carl Zeiss) and analysed using the Zeiss LSM ZEN 2012 software (Carl Zeiss).

| Statistics
Statistical significance was assessed by two-tailed Student's t test or one-way ANOVA (for multiple comparisons) using the scientific graphing analysis software (Prism, version 5.0; GraphPad Software Inc, San Diego, CA, USA). P values < 0.05 were considered statistically significant.

| Altered processing and levels of CTSs in GCD2 corneal fibroblasts
Our previous study showed that the pathogenic mutant-TGFBIp accumulates in the lysosomal compartments of GCD2 corneal fibroblasts. 13 This study indicates that lysosomes may be functionally abnormal in GCD2 corneal fibroblasts. Accordingly, we first analysed the levels of CTSs. The results showed that the levels of CTSK and CTSL were significantly reduced in GCD2 corneal fibroblasts compared to WT, but the levels of CTSB and CTSD did not differ between the two cells ( Figure 1A,B). We also measured the levels of CTSs that were in their active form. Western blot analysis showed that the active or matured forms (M) of CTSB, CTSK and CTSL were significantly decreased in GCD2 corneal fibroblasts, compared with WT ( Figure 1A,C). In contrast, the levels of CTSD did not change ( Figure 1A,B). We also analysed the ratio of the immature (IM) CTS to the active form ( Figure 1A).
The ratios of immature CTSB and CTSL to their active forms were significantly increased in GCD2 corneal fibroblasts compared with WT ( Figure 1A,D). However, the ratio of the immature forms of CTSD and CTSK to their active forms did not differ between GCD2 and WT corneal fibroblasts ( Figure 1A Confocal microscopy showed much weaker intracellular fluorescence intensity for CTSK and CTSL in GCD2 HO corneal fibroblasts, but not for CTSD ( Figure 1E,F). In addition, the mRNA expression analysis from RT-qPCR showed that the mRNA levels of CTSK and CTSL were significantly reduced, but those of CTSB and CTSD were not significantly different in GCD2 corneal fibroblasts compared with WT ( Figure 1G). Because we developed only one line of HT corneal fibroblasts, we limited our comparison between normal and pathological states to WT and HO cells, respectively. Accordingly, all data presented subsequent to Figure 1 are restricted to these cell lines.

| Perturbation of lysosomal acidification in GCD2 corneal fibroblasts
Pro-CTSs or immature CTSs are proteolytically processed to active or mature CTS forms upon acidification in lysosomes and endolysosomes. 22

| Lysosomes are highly concentrated in the perinuclear regions of the GCD2 corneal fibroblasts
The shape and distribution of lysosomes correlated with the changes in the intracellular pH. 28,29 Therefore, we investigated the distribution of lysosomes. The confocal images showed that the lysosome-associated membrane protein 2 (LAMP2), a specific lysosomal marker, was distributed in the peripheral area of the WT corneal fibroblasts ( Figure 3A).
Lysosomal distribution pattern in GCD2 HO corneal fibroblasts was highly concentrated in the perinuclear regions ( Figure 3A). Furthermore, quantification of the intensities of LAMP2 staining was significantly elevated in the HO corneal fibroblasts compared to WT ( Figure 3B).
Western blot analysis also showed that LAMP2 protein levels were significantly increased ( Figure 3C,D), whereas mRNA levels in GCD2 corneal fibroblasts did not differ compared with in WT ( Figure 3E).

F I G U R E 2
Analysis of lysosomal acidification in GCD2 corneal fibroblasts. A, Acridine orange (AO) uptake in corneal fibroblasts. Representative images of AO-stained fibroblasts that were untreated or treated with 200 nmol/L Baf A 1 (Bafilomycin A 1 ) for 4 h. Red staining is associated with acidic vesicles, whereas green staining is associated with high pH. Nucleic acids are stained green. Scale bar 20 µm. B, Altered lysosomal pH in WT and GCD2 HO corneal fibroblasts. The corneal fibroblasts were loaded with 5 µmol/L LysoSensor Yellow/Blue DND-160 for 5 min and immediately imaged under a confocal microscope. Dual-emission ratio-metric measurement of lysosomal pH using LysoSensor Yellow/Blue DND-160. These pseudo-coloured images were constructed from two emission images. In these images, the fluorescence near pH 4 was pseudo-coloured yellow-green and fluorescence near pH 7 was pseudo-coloured blue. C, Quantification of staining intensities of the LysoSensor Yellow/Blue DND-160. At least 60 corneal fibroblasts were analysed for each group. D, Western blot analysis of ATPase E levels in WT and GCD2 HO corneal fibroblasts. The total protein extracted from WT and GCD2 HO corneal fibroblasts was analysed by using anti-V-ATPase and GAPDH antibodies. GAPDH was used as a loading control. E, Quantification of relative band intensities of V-ATPase in (D). The bands were normalized against GAPDH (n = 3). Data are representative of three independent experiments. Values indicate the means ± SD. Statistical significance was determined by Student's t test. *P ≤ 0.05; **P ≤ 0.01. NS, not significant

| CTSL is the degrading enzyme of TGFBIp and exogenous CTSL expression promotes the clearance of mutant-TGFBIp
The delayed degradation of the mutant-TGFBIp within the lysosomes and the autophagolysosome (Data S1) 13

| Exogenous CTSL expression promotes the clearance of pathogenic mutant-TGFBIp
The ability of CTSL to clear mutant-TGFBIp was then investigated. In this test, to overcome the very low or lack of transfection efficiency of the corneal fibroblasts, we used a lentivirus-based gene delivery system. First, the lent-CTSL infection led to the enhancement of the CTSL in both the WT and HO corneal fibroblasts ( Figure 5A,B, lanes 2, 4, 6, 8 and 10). Second, we assayed the effects of the CTSL overexpression on the levels of the TGFBIp. Interestingly, the results showed that the mutant-TGFBIp was significantly reduced in the HO-Lenti-CTSL cells ( Figure 5A, lanes 6, 8 and 10), but not in the WT-Lenti-CTSL corneal fibroblasts ( Figure 5A, lanes 2 and 4). Previously, it has been described that apoptosis was induced in the cells expressing antisense CTSL RNA 30 and in the CTSL gene knock-out mice. 31 Accordingly, we have examined the effects of CTSs inhibition between the GCD2 and WT cells. These results showed that the cell viability of the GCD2 cells was the most susceptible to CTSL inhibition. Collectively, these results suggest that reduced CTSL with GCD2 may provide the additional cytotoxicity to pathogenic mutant-TGFBIp (Data S4).

| TFEB was reduced in GCD2, and exogenous TFEB expression rescues the lysosomal abnormalities and GCD2 corneal fibroblasts from CTSL inhibitor-induced apoptosis
Recently, the TFEB has been identified as a master regulator of lysosomal biogenesis. 26,32 TFEB regulates the expression of various autophagy-and lysosome-related proteins, including CTSs and ATG. 26,32 Accordingly, we assayed the levels and activity of TFEB. The result showed that the level of TFBE ( Figure 6A,B), the interaction between  TFEB plays an essential role in cellular homeostasis and has provided us with a novel tool to modulate lysosomal biogenesis and function, and autophagy. 24,25 Accordingly, we examined whether enhancing TFEB could recover the altered CTSs levels and lysosomal dysfunction and if it could also clear mutant-TG-FBIp. Importantly, the exogenous TFEB expression significantly promoted the reduction of mutant-TGFBIp levels ( Figure 7A,B).

| D ISCUSS I ON
We here evaluated the lysosomal roles in the intracellular accumulation of mutant-TGFBIp and concluded that the accumulation of this pathogenic mutant-TGFBIp by defective autophagy is caused by lysosomal dysfunction and that TFEB activation could be extremely important as a potential therapeutic target in treatments for GCD2.
To study the pathogenesis of GCD2, we established WT, HT and HO lines from primary corneal fibroblasts. Although we found differences between HT and WT primary corneal fibroblasts in previous study, 5,33 we were unable to determine statistical significance in this study, as only one line of HT cells was established.
Consequently, we only used data from WT and HO cells in this study to examine the differences between normal and pathological states.
Our findings demonstrate that altered maturation and reduced levels of CTSs in GCD2 corneal fibroblasts. Considering that CTSL is a TGFBIp-degrading enzyme, the accumulation of mutant-TGFBIp in lysosomes and autophagolysosomes 13 could be caused by reduced CTSL levels in GCD2 corneal fibroblasts. This is also supported by the finding that the exogenous CTSL expression levels reduced the mutant-TGFBIp in GCD2 corneal fibroblasts. Although the exact mechanisms of reduced CTSK and CTSL still need to be elucidated, we here suggest that reduced TFEB in GCD2 corneal fibroblasts may lead to altered levels of CTSs, because TFEB regulates the expression of most lysosomal enzymes, including CTSs. 26,27,32 Moreover, the exogenous TFEB expression reversed the levels of CTSL, including several CTSs. Values are expressed as the mean ± SD. Statistical tests were as follows: one-way ANOVA for (A) and Student's t test for (C). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. NS, not significant the defective autophagic clearance of pathogenic molecules. 34,35 Collectively, these data indicate that elevated lysosomal pH may be a key factor in the accumulation of mutant-TGFBIp in GCD2 corneal fibroblasts.
Lysosomal pH controls the maturation of lysosomal hydrolases and the processing of certain hydrolases, 36 including CTSs. 37 Even a minimal rise in pH is sufficient to depress lysosomal enzymatic activity and to slow down the degradation of cellular materials. 38 These studies indicate that increased immature forms of CTSB and CTSL in GCD2 corneal fibroblasts result from elevated lysosomal pH. Moreover, while the immature forms of CTSB and CTSL were elevated in GCD2 corneal fibroblasts, the maturation of CTSD and CTSK was not different between WT and GCD2 corneal fibroblasts.
These results reveal that the optimum pH of individual CTSs in activation and maturation may vary. This interpretation is also supported by previous studies that showed that the maturation of several CTSs could be affected by other CTSs 39 and by self-maturation. 30 The intracellular distribution of the lysosomes found at the perinuclear area near the microtubule-organizing centre (MTOC) and at the periphery of the cells. 31 Lysosomes are functionally influenced by their positioning in cells. 40 For example, perinuclear lysosomes can facilitate the fusions between lysosomes and autophagosomes, because these fusions primarily occur in the perinuclear area. Further, lysosomal pH is heterogeneous and affects lysosomal distribution. 29 Hence, peripheral lysosomes are more alkaline than perinuclear lysosomes. 41 Our previous study indicated that the fusions between lysosomes and autophagosomes were delayed in GCD2 corneal fibroblasts, 13 and the data from this study show that the lysosomal pH of GCD2 corneal fibroblasts is higher than that of WT corneal fibroblasts. Accordingly, these data reveal that the increased perinuclear accumulation of lysosomes could not be related to lysosomal pH in GCD2. This interpretation is supported by a study that bafilomycin A 1 treatments (as an elevator of lysosomal pH) did not alter lysosome positioning in GCD2 HO corneal fibroblasts (Data S5). Additionally, F I G U R E 6 Level and activation of TFEB were reduced in GCD2 corneal fibroblasts. A, Western blot analysis of TFEB protein levels in WT and GCD2 HO in the total cell lysate, cytosolic and nuclear fractions. β-Actin as loading controls of the total protein, 14-3-3 proteins as loading controls of the cytosolic protein, and histone H3 as loading controls of the nucleus protein were detected. WT, WT cell lysate; HO, HO cell lysate. B, Quantification of relative band intensities of TFEB in (A) (n = 3). Quantification of TFEB band intensities was normalized for β-actin, 14-3-3 and histone H3 bands, respectively. WT, WT cell lysate; HO, HO cell lysate. (C,D) Western blotting analysis of coimmunoprecipitated TFEB with 14-3-3 proteins. The extracts of the total protein were co-immunoprecipitated with anti-TFEB and anti-14-3-3 protein antibodies, respectively. The immunoprecipitates were analysed by Western blotting with antibodies against 14- We here showed a reduced level of the ATPase E subunit in GCD2 corneal fibroblasts. Although the exact mechanisms of how the V-ATPase E subunit was reduced, and whether the reduction of this E subunit can elevate lysosomal pH remain to be elucidated, the reduced V-ATPase E subunit may reveal evidence of an elevated pH in GCD2 corneal fibroblasts. This finding might be supported by a previous study demonstrating that the mutations of the V-ATPase E subunit disrupt the disassembly of the V-ATPase and affect catalytic activity. 42 Collectively, these data indicate that elevated lysosomal pH contributes to the failure of pathogenic mutant-TGFBIp degradation and CTS maturation and can render GCD2 corneal fibroblasts more vulnerable to toxic stimuli that cause further dysfunction.
The quantitative relations between protein and RNA are fundamental questions in molecular biology that are still not fully understood. In this study, the levels of LAMP2 showed significant changes at the protein level in GCD2 cells, but not at the mRNA level. We speculate that this difference may result from reduced protein turnover rather than from an increased rate of synthesis.
Because it is possible that LAMP2 could be degraded by cathepsin L, LAMP2 levels may increase as a result of a reduction in cathepsin L in GCD2 cells. This conjecture is supported by a study that shows a reduction in LAMP2 levels in conjunction with an overexpression of cathepsin L, and an increase in LAMP2 levels in the presence of cysteine cathepsin inhibitor. 3 In addition, we cannot rule out the possibility that increased lysosomal pH and reduced lysosomal activity affect the turnover of LAMP2.
In this study, exogenous CTSL expression reduced pathogenic mutant-TGFBIp levels in GCD2 corneal fibroblasts but not in WT corneal fibroblasts. In contrast, CTSL expression elevated the level of LC3-II in WT corneal fibroblasts but decreased the level in GCD2 corneal fibroblasts. Further, treatment with bafilomycin A 1 and CTSL inhibitors suggested that CTSL level is associated with autophagic fluxes. Moreover, the levels of CTSL affected only corneal fibroblasts characterized by defective autophagy or reduced CTSL. These data indicate that CTSL can clear mutant-TGFBIp by activating autophagy in GCD2 corneal fibroblasts. Accordingly, we here suggest that enhancing CTSL activity is a promising strategy for preventing and treating GCD2.
It is also possible that elevated LC3-II levels in GCD2 corneal fi- The accumulation of mutant-TGFBIp in autophagolysosome and lysosomes was sufficient to induce toxicity in corneal fibroblasts. 13,45 Accordingly, strategies for reducing pathogenic mutant-TGFBIp could provide therapeutic effects. This has been evaluated in a study in which TGFBI expression was repressed by a TGF-β signalling inhibitor 18 and clearance of mutant-TGFBIp by autophagy induction in GCD2 corneal fibroblasts. 13,16,19 Another study showed that lysosomes receive mutant-TGFBIp via the endocytic pathway in corneal fibroblasts and corneal epithelial cells. 14 However, additional studies are needed to develop this treatment strategy. Recent studies suggest a potential role for TFEB as a new therapeutic target for diverse diseases that are linked to impaired autophagy and lysosomal function. Therefore, we hypothesized that TFEB activity is involved in lysosomal abnormalities, and enhancement of TFEB can rescue the impaired autophagy-lysosome pathway of GCD2 corneal fibroblasts. In support of this hypothesis, we found that TFEB activation and expression were reduced in GCD2 corneal fibroblasts. Furthermore, most importantly, exogenous TFEB expression not only promoted autophagosome formation (as shown by elevated LC3-II levels) but also restored lysosomal function (as shown by up-regulated CTSK and CTSL expression), which ultimately restored the disrupted lysosomal dysfunction.
Besides autophagy-lysosomal systems regulation, TFEB participates in various biological functions. 46 The present study also showed the protective effects of TFEB (such as the reduced cleavage of PARP1) against the apoptosis of GCD2 corneal fibroblasts induced by CTSL inhibitor. These findings suggest that the activation of TFEB could protect the degeneration of corneal fibroblasts of GCD2 patients. However, it is currently unknown how TGFBI mutation influences the level of TFEB. There is increasing evidence to support that expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α) co-activates the expression of TFEB. 47 Recently, it was reported that PGC-1α can robustly induce the expression of TFEB and rescue proteotoxicity in a mouse model of Huntington's disease. 48,49 These studies showed that PGC-1α can rescue the impaired TFEB induction and significantly reduce TFEB expression by PGC-1α reduction. Another study showed that PGC-1α expression directly parallels the level of TFEB. 50,51 In addition, TFEB translocation from the cytosol to the nucleus was impaired in the absence of PGC-1α. 52 Although it remains unclear whether the pathogenic mutation causes a lossof-function or gain-of-function of TGFBIp, Maeng et al showed that TGFBIp activates focal adhesion kinase (FAK) signalling. 53 Furthermore, a recent study showed that phosphorylation of FAK was enhanced by TGFBIp and was suppressed significantly by an integrin α5β1 inhibitor. 54 Considering that TGFBIp interacts with several integrins 55 including α5β1, 56 these data indicate that TGFBIp is an upstream regulator of the integrin/FAK/PGC-1α signalling pathway. Accordingly, TFEB in GCD2 corneal fibroblasts can be reduced by reduced integrin/FAK/PGC-1α signalling. Therefore, we here predict that PGC-1α is reduced in GCD2 corneal fibroblasts and thereby reduces the level of TFEB. Collectively, TFEB is a potential therapeutic target for maintaining the physiological function of corneal fibroblasts from the dysfunction of autophagy and lysosomes. Preclinical studies are also needed to evaluate the consequence of TFEB overexpression or activation as a candidate therapeutic target for treating GCD2.