Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.


Introduction
The trabecular meshwork (TM) is a tissue located in the anterior segment of the eye, which has a major role in regulating intraocular pressure (IOP), by controlling the resistance to aqueous humor (AH) outflow from the eye. Functional failure of the TM causes ocular hypertension and predisposes the development of primary open angle glaucoma (POAG), a group of eye diseases leading cause of permanent blindness worldwide [1,2].
The TM consists of sheets of connective tissue beams through which AH must flow before exiting the eye. Each beam is composed of a central elastic core surrounded by collagen fibers embedded in a ground substance, lined by TM endothelial cells. With aging and in disease, the trabecular beams become thicker due to thickening of the basement membrane underlying the trabecular cells or changes in the extracellular matrix (ECM) within the central core. In the TM of glaucomatous eyes, deposition of long-spacing collagen is also frequently observed [3]. The exact causes responsible for this thickening of the beams and deposit of ECM remain unknown, but are likely due to excessive ECM synthesis, decreased proteolytic degradation, or both [4]. Excessive synthesis possibly plays a role because the aqueous humor (AH) of patients with POAG contains elevated concentrations of TGF-β2 [5,6], a cytokine that promotes ECM deposition and increases outflow resistance [6,7]. Degradation is controlled extracellularly by the action of proteases. Regulation of the ECM composition in the TM is known to play a major role in both outflow pathway physiology and pathophysiology [4,8,9]; however, the exact mechanisms underlying ECM remodeling with IOP changes and those associated with its alteration in glaucoma remain poorly understood [10].
Despite the general belief that matrix metalloproteinases (MMPs) are the only group of enzymes responsible for ECM degradation, emerging evidence in other tissues suggests that other proteases or the coordinated action of several types of proteases, other than just MMPs, participate in bulk matrix degradation and ECM remodeling [11]. In this line, previous studies conducted in our laboratory reported for the first time a role of the lysosomal cysteine protease cathepsin B (CTSB) in intracellular degradation of ECM components in TM cells [12]. We found partially degraded ECM products associated with active CTSB in lysosomal compartments. Moreover, CTSB was found to be constitutively expressed in the cell surface and secreted to the culture media in its inactive pro-form. CTSB was also detected in AH samples. Surface expression and secretion of cathepsins are not unique to TM cells. Although preferentially located in lysosomes, specific subtypes of cathepsins (CTSB, CTSL, CTSK, CTSD) have been located on the cell surface as well as secreted into the extracellular space in different cell types, preferentially of mesenchymal origin, either constitutively or under stress conditions [13][14][15]. In particular, surface CTSB has been localized in caveolae in HUVEC endothelial and colorectal carcinoma cells [16,17]. Caveolae are lipid-rich invaginations of the plasma that play a key role in regulating cell signaling, lipid homeostasis, and adaptation to membrane tension. Emerging studies are now showing that caveolae are also important regulators of ECM remodeling, acting as focal proteolytic degradation sites. CTSB activity at the cellular membrane has been associated with the pericellular degradation of ECM and basement membrane proteins and contribute to cell migration in cancer cells [15,17,18]. Interestingly, CTSB has been also shown to promote epithelial-mesenchymal transition (EMT) in tumor cells and contribute to tissue fibrosis [19].
Given the relevance of fibrosis and EMT transition in outflow pathway pathophysiology, we decided to follow up on our initial observations and to further investigate a role of CTSB in ECM remodeling and TM tissue function. Here, we show for the first time that surface CTSB localizes in the caveolae and participates in the pericellular proteolytic cascade in TM cells and TGFβ signaling.

Cell Culture
Primary human TM cells were isolated from dissected TM tissue from discarded corneal rims after surgical corneal transplantation at Duke University Eye Center and maintained as described earlier [20]. Cells were passaged when confluent at a 1:2 ratio. Cells from passage 4-8 were used in this study. HTM cells were characterized by morphology and by the upregulation of myocilin in response to dexamethasone treatment, in accordance to the consensus recommendations for TM cell isolation, characterization, and culture [21]. The protocols involving the use of human tissue were consistent with the tenets of the Declaration of Helsinki.

Preparation of Caveolae-Enriched Fractions
Caveolae-enriched fractions were prepared using the Caveolae Isolation Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer's instruction. This kit is based on the resistance of caveolae to solubilization in ice cold Triton X-100. Briefly, one confluent 100 mm-tissue culture dish of hTM cells was washed twice with cold 1 × PBS and lysed on ice in proprietary Lysis Buffer containing 1% Triton X-100. Fractions were fractionated by OptiPrep density gradient ultracentrifugation and collected in 1 mL fractions. Caveolin-1 (Cav1) was used as a marker for caveolae-enriched fractions (fractions 2-5).

Whole-Cell and Tissue Lysates Preparation
Cells and dissected iridocorneal region tissue were washed twice in PBS and lysed for 30 min in RIPA buffer (Millipore Sigma, Burlington, MA, USA; R0278) containing 1:100 Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA; 78842). After sonication for one minute on ice, lysates were clarified by centrifugation. Protein concentration was determined using a protein assay kit (Micro BCA, Thermo Scientific, Waltham, MA, USA; 23235).

Western Blot Analysis
For WB analysis, proteins (2.5-10 µg), cell culture medium (3.5-20 µL), or caveolaenriched fractions (15 µL) were separated by SDS-PAGE (10-15%) and transferred to PVDF membrane (Bio-Rad). Membranes were blocked with 5% non-fat dry milk in PBS-T (0.1% Tween-20) for 1 h at room temperature or overnight at 4 • C and incubated with primary antibodies diluted in blocking buffer overnight at 4 • C. Membranes were washed three times with PBS-T and incubated with peroxidase-conjugated secondary antibodies for 1-2 h at room temperature. Bands were detected using chemiluminescence substrate (HyGlo Quick Spray, Denville, Swedesboro, NJ, USA; E2400). Blots were scanned using ChemiDoc Image System and analyzed by densitometry using Image J. ACTB or GAPDH were used for normalization. Primary antibodies and dilutions used in the study are listed in Table 1. Total RNA was isolated from human TM cells using RNeasy kit (Qiagen) according to the manufacturer's protocol and treated in-column with DNase I (Qiagen). RNA yields were measured using Nanodrop spectrophotometry (NanoDrop 1000, Thermo Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from total RNA (1 µg) by reverse transcription using oligo(dT) primer and reverse transcriptase (SuperScript First Strand System, Thermo Scientific, Waltham, MA, USA). Real-time PCR was performed in a 20 µL mixture containing 1 µL of the cDNA preparation diluted five times, 10 µL master mix (SsoFast EvaGreen Supermix, Bio-Rad, Hercules, CA, USA), and 500 nm of each primer in a thermocycler system (iCycler iQ; Bio-Rad, Hercules, CA, USA) using the following PCR parameters: 95 • C for 5 min, followed by 50 cycles of 95 • C for 15 s, 60 • C for 15 s, and 72 • C for 15 s. The fluorescence threshold value (Ct) was calculated using the thermocycler system software. The absence of nonspecific products was confirmed by both the analysis of the melt curves and by electrophoresis in 3% acryl-agarose gels. β-Actin and GAPDH served as an internal standard of mRNA expression. The change (x-fold) was calculated with the formula 2 −∆∆Ct , where ∆Ct = Ct gene − Ct Act , and ∆∆Ct = ∆Ct Exp − ∆Ct Con . The sequences of the primers used for the amplifications are: PAI-1: TGF TGG TGA ATG CCC TCT ACT and CGG TCA TTC CCA GGT TCT CTA.

ELISA
Secreted TGFβ1 and TGFβ2 were measured by ELISA (Invitrogen, BMS249-4 and BMS254, respectively) in 100 µL of clarified culture media, according to the manufacturer's protocol.

CTSB ko Mice
CTSB ko mice were obtained from Dr. Henriette Van Praag at Florida Atlantic University (Boca Raton, FL, USA) [23]. Generation of these mice has been described in [24]. Mice were maintained and aged in situ. Genotyping was performed by PCR in digested tail genomic DNA using the following primers with a temperature annealing of 57.5 • C for 45 s (F: GGT TGC GTT CGC TGA GG; R: AAC AAG AGC CGC AGG AGC). CTSB ko and littermates CTSB wild-type (males and females) were used for experimental purposes. Animals were maintained under a 12 h light/dark cycle, fed a standard mouse diet, and provided with water ad libitum. Animal euthanasia was performed via CO 2 asphyxiation, followed by bilateral thoracotomy, prior to immediate enucleation. Enucleated eyes destined for electron microscopy (EM) were fixed under perfusion in 2% glutaraldehyde/0.1 M cacodylate buffer. Tissues destined for WB analysis were immediately dissected and flash frozen on dry ice. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Duke University and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

IOP Measurements
IOP was measured between 10 a.m. and 12 p.m., bi-monthly in isoflurane-anesthetized mice using a TonoLab rebound tonometer (Colonial Medical Supply, Franconia, NH, USA). Six measurements, taken within 5 min after anesthesia, were collected per eye and averaged to obtain a single IOP value per eye for each session.

Ex-Vivo Outflow Facility
Eyes were enucleated within 5 min of death and kept in a warmed solution of 5.5 mM glucose in Dulbecco's PBS with calcium and magnesium (DBG) until perfusion (15-20 min). Outflow facility measurements were conducted ex-vivo using the iPerfusion MK V system [25]. For this, eye cups were affixed with a small amount of cyanoacrylate glue (Locktite, Henkel Corporation, Rocky Hill, CT, USA) to platforms within heated baths filled with DBG. XYZ micromanipulators (World Precision Instruments, Sarasota, FL, USA) were used to cannulate the anterior chamber of each eye with a glass microneedle. The glass microneedles were manufactured in-house by pulling glass capillary tubes (PN-3 Puller, Narishge Scientific Instrument Lab, Tokyo, Japan) and sharpened to a 20 degree bevel (BV-10, Sutter Instrument Company, Novato, CA, USA). An initial pressure of 8 mmHg for a period of 30 min was applied to allow acclimatization of the eyes to pressure and temperature environment. A nine-step post acclimatization protocol was conducted with applied pressures of 5.0, 6.5, 8.0, 9.5, 11.0, 12.5, 14.0, 15.5, and 17.0 mmHg before returning to 8.0 mmHg. Outflow facility was calculated with the iPerfusion integrated software [25].

Electron Microscopy (EM)
Enucleated eyes were post-fixed for 24 h in 2% glutaraldehyde/0.1 M cacodylate buffer solution and transferred to cold 3% glutaraldehyde/0.1 M cacodylate buffer for 2 h. Eyes were then washed in cacodylate buffer, post-fixed in 2% OsO4/0.1 M cacodylate, and dehydrated though increasing ethanol gradient ending in two cycles of propylene oxide. Tissue was infiltrated by immersion in a 1:1 mixture of propylene oxide and Epon 812 resin under a vacuum for 4-10 h. Labeled molds were then heated at 68 • F for 8-10 h. Sections were cut at 65 nm thick using a Leica EM CU7 and contrast stained with 2% uranyl acetate/4% lead citrate solution. Ultrathin sections were visualized on a JEM-1400 transmission electron microscope (JEOL) using an ORIUS (1000) CCD camera. Electron microscopic images of trabecular meshwork at lower magnification (X3000-4000), and higher magnification (X10000-12000) from four wide-type and three CTSB KO mouse eyes were analyzed and compared.

Statistical Analysis
All in vitro experimental procedures were repeated at least three times in independent experiments with hTM primary cultures from different donors. Animal studies were conducted with n = 4 to 11, as indicated for each particular experiment. Data were presented as the mean ± S.D. Statistical analysis was done in GraphPad Prism software using Student's t-test or 2-way ANOVA with Tukey post-test for multiple comparisons. p < 0.05 was considered to be statistically significant.

Cathepsin B Localizes in Caveolae with Components of the Proteolytic Cascade and Is Secreted into the Culture Media in Primary Cultures of TM Cells
Our laboratory previously reported that CTSB is constitutively expressed at the cell surface in porcine TM cells and secreted into the culture media in its inactive pro-CTSB form [12]. We want to investigate whether membrane CTSB localizes to caveolae, as described in HUVEC endothelial and human colorectal carcinoma cells [16,17]. For this, we purified caveolae-enriched fractions from primary human TM cultures and evaluated CTSB expression in the collected protein fractions by WB. We first analyzed the expression of the caveolae marker, caveolin 1 (CAV1). As seen in Figure 1A, CAV1 was mostly found in the low-density fractions 2 through 5, hereafter designated as caveolae fractions, with a sharp decreased in high-density fraction 6. Excitingly, pro-CTSB and active single-chain CTSB (scCTSB) were detected in the caveolae-enriched fractions 4-5 and 5, respectively. It should be noted that double-chain CTSB (dcCTSB) was found in whole-cell lysates (CL) and fraction 6, but it was not localized in the caveolae-enriched fractions. scCTSB to dcCTSB transition occurs in the lysosomes; therefore, its expression is not anticipated in the caveolae. It should also be noted that the caveolae purification method is based on the unique resistance of caveolae to solubilization in ice-cold Triton X-100. While caveolae do not solubilize, endosomes and lysosomes (potential contaminants) do [26]. We additionally investigated the presence in the caveolae of components of the proteolytic cascade urokinase plasminogen activator (uPA), its receptor (uPAR), and that of annexin A2 (ANXA2), a proposed binding partner of CTSB and the cell membrane. As observed in Figure 1B, all of them (uPA, uPAR and ANXA2) were identified in the caveolae-enriched fractions. A band of~55 kDa immunoreacting with anti-uPA antibody, referred in the literature as uPA bound to plasminogen activator inhibitor type-1 (PAI-1), its natural inhibitor, was also identified. We next confirmed whether, as we previously reported in porcine TM cells, CTSB was secreted by human TM cells. For this, we analyzed CTSB expression in conditioned media from cultured human TM cells; uPA was used as control. Similar to our previous findings, pro-CTSB, but not active CTSB, was detected ( Figure 1C). Altogether, these data strongly indicate that CTSB localizes in the caveolae, together with components of the proteolytic cascade, and it is secreted into the culture media in primary cultures of human TM cells.

Cathepsin B Mediates Pericellular Proteolysis of ECM in TM Cells
The finding that CTSB localizes in caveolae with other members of the proteolytic cascade opens the question of whether CTSB participates in the pericellular degradation of ECM at the cell surface. This was investigated by in situ live zymography using DQ-gelatin, a highly quenched fluorescein-labeled gelatin that becomes fluorescence upon proteolytic digestion. Human TM cells were grown onto gelatin containing DQ-gelatin (25 µg/mL). Proteolytic degradation of DQ-gelatin was monitored using the Celena X imaging system. Degradation products of DQ-gelatin were observed at the cellular periphery after 48 h of culture (Figure 2A, green fluorescence, red arrows). We next repeated the experiment in the presence of the CTSB or the cysteine protease inhibitors, Ca074Me (40 µM) and E64d (10 µM), respectively. As seen in Figure 2B, reduced gelatinolytic activity (decreased green fluorescence) was observed at the pericellular region in the presence of the inhibitors. We additionally quantified by flow cytometry the effect of Ca074Me, E64d, and marimastat, an MMP inhibitor, on the intracellular degradation of DQ-gelatin and DQ-collagen I ( Figure 2C). Similar to our results in porcine TM cells [12], degradation of DQ-substrates was significantly blocked by inhibiting CTSB (gelatin: 30.05 ± 16%, p < 0.01, n = 3; Col I: 58 ± 17%, p < 0.01, n = 3) and cysteine cathepsin activities (gelatin: 33 ± 15%, p < 0.05, n = 3; Col I: 67 ± 19%, n = 3), at same levels of those observed with the MMP inhibitor (gelatin: 25 ± 15%, p < 0.01, n = 3; Col I: 58 ± 16%, p < 0.05, n = 3). Together, the results strongly confirm a role of CSTB and cysteine cathepsins in the pericellular and intracellular degradation of ECM components.

Inhibition of CTSB Upregulates and Increases the Secretion of Plasminogen Activator Inhibitor 1 (PAI-1)
Several studies have shown the ability of CTSB to degrade ECM either intracellularly, extracellularly, or both by initiating a proteolytic cascade that involves uPA, plasminogen (PLG), and PAI-1 [11,14,17,[27][28][29][30][31]. We tested the effect of CTSB inhibition on the expression levels of the components of the proteolytic cascade PLG, uPA, and PAI-1 by WB. For this, we treated three independent primary cultures of human TM cells with Ca074, Ca074Me, or E64d for 24 h. Intriguingly, inhibition of cysteine cathepsins or CTSB activity significantly increased the protein levels of PAI-1 ( Figure 3A,B; Ca074Me: 250 ± 27%, p = 0.0007; E64d: 242 ± 58%, p = 0.013, t-test, n = 3). No significant changes in the protein levels of PLG or uPA were observed (not shown). We additionally confirmed these results in human TM with silenced expression of CTSB. Cells were transfected with siRNA targeting CTSB (siCTSB) or control siRNA (siNC) for 72 h and treated with TGFβ2 (10 ng/mL, 48 h). TGFβ2 is a known upregulator of PAI-1 and was used as a positive control. PAI-1 protein levels were evaluated by WB in whole cell lysates and conditioned media; mRNA PAI-1 levels were quantified by qPCR. As seen in Figure 3C-E, siCTSB knockdown significantly increased both intracellular and secreted protein levels of PAI-1. Higher PAI-1 mRNA levels were also observed in siCTSB-transfected cultures ( Figure 3F).

CTSB Is Secreted into the Culture Media in Response to Cyclic Mechanical Stretch
Cells in the TM are exposed to mechanical stretch due to continuous changes in IOP. It is well-known that TM cells sense and respond to those mechanical forces by eliciting a number of responses, among them ECM remodeling, which are believed to be directed at maintaining IOP homeostasis [32,33]. We wanted to check whether mechanical stretch could affect expression or secretion of CTSB. For this, we subjected three primary cultures of human TM cells to cyclic mechanical stress (8% elongation, 24 h). CTSB expression was evaluated in whole cell lysates and culture media by WB. As seen in Figure 4A and quantified in 4B, a decrease in intracellular CTSB (single-and double-chain forms) was found in the stretched cultures (sc-CTSB: 0.68 ± 0.39 folds; dc-CTSB: 0.39 ± 0.34 folds, p < 0.05, n = 4). In contrast, higher levels of CTSB (pro-CTSB) were observed in mechanically stretched cells.

CTSB Knockout Mice Display Defective uPA Activation in the Iridocorneal Region
Next, we investigated the potential effects of CTSB deficiency in outflow pathway tissue physiology. For this, we monitored IOP and outflow facility in CTSB deficient mice (CSTB ko ) and littermate CTSB mice. As seen in Figure 6A, no difference in IOP was observed between CTSB and CTSB ko mice, evaluated up to 18-month-old age. Interestingly, an increased in outflow facility was observed in CTSB ko mice compared to CTSB control, although it did not reach statistical significance ( Figure 6B, CTSB ko : 5.155 ± 1.836 nl/min/mmHg vs. CTSB: 4.03 ± 0.739 nL/min/mmHg, p = 0.07, t-test, n = 10). Based on the findings in our in vitro studies, we analyzed the expression levels of several components of the proteolytic cascade in dissected iridocorneal region tissues of CTSB ko and littermates CTSB, based on antibody availability ( Figure 6C, D). Absence of CSTB in the iridocorneal region tissue in CTSB ko mice was confirmed. Compared to CTSB, CTSB ko mice displayed a very significant increase in pro-uPA (CTSB ko : 1.850 ± 0.178 RDU vs. CTSB: 0.735 ± 0.494 RDU; p = 0.005, t-test, n = 4). This was accompanied by a decrease in uPA-PAI-1 levels (CTSB ko : 0.248 ± 0.167 RDU vs. CTSB: 0.610 ± 0.198 RDU; p = 0.006, t-test, n = 6). Decreased levels of cystatin C (CSTC), an endogenous CTSB inhibitor, were also found in KO mice (CTSB ko : 0.525 ± 0.215 RDU vs. CTSB: 0.838 ± 0.279 RDU; p = 0.04, t-test, n CTSB = 7, n CTSBko = 6). Additionally, CTSB ko mice displayed higher levels of the fibrotic marker SMA (CTSB ko : 1.255 ± 0.398 RDU vs. CTSB: 0.738 ± 0.229 RDU; p = 0.016, t-test, n CTSB = 7, n CTSBko = 6), but no significant changes in fibronectin (FN1) were detected. EM morphometric analysis showed no gross changes in outflow pathway morphology in CTSB ko mice (Figure 7). Both cells and ECM look normal in the TM of KO mice compared to CTSB mouse eyes; no obvious differences were observed in either the endothelial cell of Schlemm's canal, juxtacanalicular connective tissue regions, or TM endothelial lining cells between the two groups.

Discussion
In this manuscript, we report for the first time that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in vitro and in vivo in CTSB ko mice.
Caveolae are unique invaginated lipid-rich regions of the plasma membrane highly abundant in mechanically stretched cells that play a major role in regulating cell signaling, lipid homeostasis, and adaptation to membrane tension. Although less investigated, emerging studies are showing that caveolae are important regulators of ECM remodeling, acting as focal proteolytic degradation sites. Disruption of caveolae formation in Cav1-deficient cells results in abnormal expression of matrix proteins [36][37][38]. In TM cells, Cav1 deficiency has been shown to alter the physiological catabolism of ECM and modulate outflow resistance [39]. First, many proteases implicated in ECM remodeling are associated or localized to the caveolae. Second, caveolae mediates internalization, via endocytosis, of partially degraded ECM fragments and targets them for final degradation within lysosomes. Along these lines, our studies revealed the localization of members of the proteolytic cascade, pro-uPA and uPAR, along with that of the cysteine cathepsin CTSB in Cav-1-enriched fractions from TM cells. Interestingly, AnxA2, a reported cell surface receptor for tPA and CTSB was also localized in the caveolae [28]. CTSB in the caveolae was found in its inactive (pro-CTSB) and as a single chain (sc-CTSB) forms, consistent with the notion that sc-to dc-maturation occurs in lysosomes. An advantage of localization of CTSB in the caveolae is the quick availability. Our results showed increased secretion of pro-CTSB in response of mechanical stretch. It is plausible that mechanical deformation of caveolae with mechanical forces triggers the release of the pro-enzyme and be one of the contributing factors initiating ECM remodeling in stretched cells.
Using a live-cell proteolysis assay in combination with CTSB inhibitors, we confirmed the contribution of cell surface CTSB in the pericellular degradation of ECM components. ECM degradation at the cell surface occurs by the coordinated action of different types of proteases and their receptors. In a proposed model for tumor cells, activation of receptorbound-pro-CTSB (AnxA2/CTSB) at the caveolae triggers the uPA/plasminogen/plasmin proteolytic cascade that culminates with MMP activation and ECM breakdown. CTSB can also directly activate some MMPs. Very interestingly, pharmacological inhibition or silencing of CTSB resulted in transcriptional upregulation and increased secretion of PAI-1 in TM cells. PAI-1 is a member of the superfamily of serine-protease inhibitors or serpins and potent inhibitor or tPA and uPA. PAI-1 binds to uPA/uPAR complex and targets uPAR for degradation. To our knowledge, this is the first time that regulation of PAI-1 expression by CTSB is reported and adds a novel mechanism by which CTSB can modulate ECM proteolysis in addition to proteolytic activation. Consistent with these results, we observed increased levels of pro-uPA in the dissected iridocorneal mouse tissue of CTSB ko mice compared to CTSB controls. CTSB ko mice also displayed lower levels of CTSC, an endogenous inhibitor for CTSB, suggesting a regulatory feedback between the two proteins.
Noteworthy are the increased levels of SMA in the iridocorneal region of CTSB ko mice. A marked increase in SMA protein levels was also identified in vitro in hTM cells treated with the CTSB inhibitor Ca074Me. One could argue that the observed increase in pSmad2/3 levels could explain the increase in SMA. However, silencing CTSB expression elevated SMA levels in just two out of the four cell strains tested, while elevated pSmad2/3 was observed in all of them. Intriguingly, the capability of CTSB to modulate SMA seems to be a cell strain-intrinsic factor, since independently conducted replicate experiments using the same cell strains yield the same outcomes. It is plausible that elevated SMA levels might also result from defective CTSB-mediated degradation of either SM or SMA negative transactivation signal. That might partially explain the why irreversible inactivation of CTSB activity with Ca074Me resulted in such remarkable increase in SMA protein levels. This, however, requires further investigation.
A role of CTSB in EMT and tissue fibrosis has been investigated in a very recent work [34]. In this elegant study, Zhang et al. report a differential role of cysteine cathepsins in TGFβ signaling and kidney fibrosis. Interestingly, using a model of unilateral ureteral obstruction, the authors found that CTSB or CTSL deficiency in mice exacerbates kidney fibrosis, whereas CTSS or CTSK deficiency protects it. Similar to our own results, they detected higher expression levels of SMA in CTSB ko mice. More, also as in siCTSB-transfected TM cells, tubular epithelial cells deficient in CTSB showed increased pSmad2/3 in response to TGFβ treatment. Intriguingly, they found that this resulted from the interaction of membrane surface CTSB with TGFBR1, targeting TGFBR1 for degradation and abolishing downstream TGFβ signaling. CTSB has been also shown to regulate TGFβ pathway by participating in the intracellular proteolytic activation and maturation of TGFβ1 [35]. Although not confirmed in TM cells, this could certainly explain the observed lower secreted levels of TGFβ1 and TGFβ2 in siCTSB-transfected TM cells.
Fibrosis and EMT transition have been linked to the pathogenesis of glaucoma. The fact that despite the increase in SMA and diminished pro-UPA activation CTSB ko mice showed a trend in higher-than rather lower-outflow facility and no significant morphological or physiological changes in the outflow pathway tissues was somehow a little disappointing and unexpected. As we previously reported, glaucomatous TM cells display lower CTSB protein levels [40]. Several contributing factors can explain these results. First and foremost is the known compensatory role of cysteines cathepsins, in par-ticular between CTSB and CTSL [41]. The second factor is age. While no differences in IOP were noted over time, outflow facilities and morphological analysis were conducted just in young mice. The third factor is TGFβ. As indicated by our results presented here and those in [34] [35], there is a complex relationship between CTSB and TGFβ signaling. While decreased levels of TGFβ1 and TGFβ2 were noted in TM cells with knockdown expression of CTSB, the concentration of TGFβ2 in AH from glaucomatous eyes is known to be higher than that in age-matched control eyes. It is therefore expected that effects of CTSB differ in a physiological compared to a pro-fibrotic environment. Future experiments will be directed at exploring the effect of aging and pro-fibrotic environment in CTSB-mediated activation of the proteolytic cascade and ECM remodeling.

Conclusions
In summary, we have identified here CTSB as a novel mechanosensitive player regulator of the proteolytic cascade and TGFβ signaling in TM cells. Small drugs targeting cathepsins are currently being developed and tested in clinical trials to treat diseases with imbalance ECM turnover. Similar approaches targeted at enhancing CTSB activity or restoring CTSB levels in the glaucomatous TM could be developed to attenuate fibrosis and ECM deposition in the TM in glaucoma.

Supplementary Materials:
The following are available online at https://www.mdpi.com/2077-038 3/10/1/78/s1, Figure S1: Effect of CTSB deficiency on SMA. Protein levels of SMA in whole-cell lysates (5 µg) from four independent hTM strains transfected with siCTSB or siNC for 72 h and subjected to TGFβ2 (10 ng/mL) or TGFβ2 (10 ng/mL) treatment for 48 h. Graph represents relative protein levels calculated from densitometric analysis of the bands.