A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing

The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student’s t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.

Introduction cells (in vitro models) are frequently used in screening/discovery studies. The major disadvantage of using TM cells is that the IOP and GC responsiveness of the eye from which the TM cells are isolated is usually unknown. Without this information, it is difficult to verify whether a TM cell strain is a responder or non-responder. The lack of GC responsiveness information may explain the inconsistency between several microarray studies [31][32][33][34][35][36].
In this study, we combined our bovine ex vivo and in vitro models to determine the genes that are differentially expressed in bovine TM (BTM) cells. Our study is unique because: 1) we used TM cells from eyes with known GC responsiveness and 2) we used RNA sequencing (RNAseq) to compare gene expression between GC responders and non-responders.

Bovine Anterior Segment Perfusion Culture
Paired bovine eyes were obtained from a local abattoir and transported to the laboratory on ice within six hours from time of sacrifice. One eye from each pair was subjected to ex vivo perfusion organ culture, while the fellow eye was used to establish the BTM cell strain. The perfusion culture procedure was previously described [29]. Briefly, the extraocular tissue was removed from the eyes, and the eyes were sterilized with Betadine (Purdue Products, Stamford, CT) for 2 minutes, followed by two rinses with PBS. The eyes were scored and dissected with scissors along the equator, and the posterior segment was discarded. We carefully removed the vitreous, uveal tract, and lens without disturbing the TM. The remaining anterior segment was then mounted on a custom made Plexiglass dish. A size-matched Plexiglass Oring was then used to clamp the anterior segment against the Plexiglass dish at the equator with four plastic screws. This mounting created a water-tight artificial anterior chamber. Each Plexiglass dish had two embedded cannulas: one for medium infusion and the other for IOP measurement via a pressure transducer (ADInstruments, Colorado Springs, CO). DMEMhigh glucose medium (Thermo Scientific, Waltham, MA) containing 1% glutamine, 1% penicillin + streptomycin, and 1% amphotericin B (Sigma-Aldrich, St. Louis, MO) was infused at a constant infusion rate of 5μL/min using a syringe pump (PHD2000; Harvard Apparatus, Holliston, MA). Pressure transducers were connected to a data acquisition system (PowerLab; ADInstruments) consisting of a signal amplifier, a bridge amplifier, and a computer with the LabChart software (ADInstruments). Stable baseline IOPs were established within the first 24 hours of bovine anterior segment perfusion. Eyes were then treated with 100 nM DEX initially dissolved in ethanol (EtOH) (Sigma-Aldrich). IOPs were recorded every minute. For data analysis, baseline IOP was defined as the average of IOP measured 12 hours prior to treatment. After treatment started, IOP was averaged every 24 hours. ΔIOP was defined as averaged IOP minus baseline IOP. Maximum ΔIOP (mΔIOP) was the highest IOP post-treatment and was used to determine DEX-responsiveness. Eyes with an mΔIOP equal or greater than 2.82 mmHg were considered responders and those below 2.82 mmHg were considered non-responders as previously described [29].

Establishment of BTM Strains & Assessment of DEX Responsiveness
The contralateral eye of paired bovine eyes was used for BTM cell isolation and cell culture. The TM tissue was dissected under a dissection microscope, and placed into the well of a 24-well culture plate filled with DMEM-high glucose medium (Thermo Scientific) containing 1% glutamine, 1% penicillin + streptomycin, and 10% fetal bovine serum (FBS) (AtlasBiologicals, Fort Collins, CO). BTM cells migrated from the TM tissue onto the culture plate surface within a few days. BTM cells were passaged 1:2 to 1:3 from a 24-well plate to 12-well plate, 6-well plate, and T-25 flask. Confluent BTM cells were then treated with either 0.1% EtOH as a vehicle control or 100nM DEX for 7 days. To verify the identity of these TM cells, the formation of cross-linked actin networks (CLANs) as well as the expression of TM cell markers, including collagen IV, laminin, and α-smooth muscle actin were verified in all the BTM cells strains using immunofluorescent staining [37][38][39]. Conditioned medium was collected and used for Western immunoblotting (WB). After electrophoresis using 4-15% SDS-PAGE and transfer, the blots were blocked with 5% dry milk and incubated with the rabbit anti-fibronectin antibody (Millipore, Billerica, MA). The blots were washed and incubated with a secondary goat anti-rabbit antibody conjugated with HRP (Cell Signaling Technology, Danvers, MA). SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used for signal detection and images were taken using the FluroChem TM 8900 imager (Cell Biosciences, Santa Clara, CA). Some SDS-PAGE gels were stained with Coomassie blue (GelCodeBlue, Thermo Scientific) to ensure equal protein loading.

RNA Extraction
Confluent BTM cells cultured in 6 well plates from established strains were treated with EtOH or DEX as previously described in DMEM+0.5% FBS for 7 days, and medium was changed every other day. Total RNA was extracted using the RNA purification kit (RNeasy Mini Kit, Qiagen) with DNase I treatment for 15 minutes. RNA was quantified using the NanoDrop 2000 (Thermo Scientific) and RNA integrity (RIN) was measured using the Agilent Bioanalyzer (Agilent Technologies). Bovine TM cell strains with an RIN >9.5 and a concentration of 100ng/μl or higher were used for RNA sequencing.

Expression Profiling by RNASeq
Transcript profiling using the RNASeq was performed at the University of Iowa, Iowa Institute of Human Genetics, Genomics Division (Iowa City, IA). Briefly, 300 ng total RNA was sheared using the Covaris E220 (Covaris, Inc., Woburn, MA), converted to cDNA and ligated to sequencing adaptors containing indexes using the Illumina TruSeq stranded mRNA sample preparation kit (Cat. #RS-122-2101) following the manufacturer's recommended protocol (Illumina, Inc., San Diego, CA). The resulting libraries were normalized by index, pooled, and sequenced using Illumina v3 (2 x 100 bp paired-end) sequencing chemistry run on an Illumina HiSeq 2000 (Illumina, Inc.). The data have been submitted to http://www.ncbi.nlm.nih.gov/sra (accession number PRJNA315985).

Mapping and Expression Quantification
The UMD3.1 genome assembly of bovine was used for mapping and annotation. We used Tophat2 [40] to perform mapping, Cuffquant for quantitation, and Cuffnorm and Cuffdiff for normalization and differential expression analysis. The 10 th percentile of the level of expression was added to the fragments per kilobase of exon per million fragments mapped (FPKM) values reported by Cuffdiff to regularize the expression values. This diminishes artifacts of large or small fold change values as a result of a measured value for expression being close to zero.
To determine if the genes were both statistically and biologically significant, we performed a two-step analysis: 1. Selected the genes with the FDR-adjusted p-value (i.e. the q-value) < 0.05.
2. For the genes that already showed statistical significance (p<0.05), we further set a cutoff line of 20% change, i.e. only genes with !20% increase or decrease in expression were selected.
For DEG group #3, we performed a 3 rd analysis. We compared the increase or decrease between responders and non-responders. Only genes showing !20% difference in increase or decrease between responder vs. non-responders groups are listed. We included an example in S1 Appendix to clarify our analyses.

Real Time qPCR
The same RNA samples used for RNAseq were used for reserve transcription. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). In each qPCR test tube, 20 μl reaction mix was prepared using SSoAdvanced SYBR Green Supermix (Bio-Rad). qPCR was performed in the CFX96 thermocycler (Bio-Rad). The thermoprofile was 40 cycles of 95˚C for 10 seconds and 60˚C for 30 seconds, followed by a dissociation curve check. The PCR primers are listed in Table 1. The ΔΔCt method was used to calculate gene expression changes, and actin was used as an internal control.

Pathway and Protein-protein Interaction Analysis
Pathways associated with genes identified by RNAseq were analyzed using the WEB-based GEne SeT AnaLysis Tool kit (WebGestalt) (http://bioinfo.vanderbilt.edu/webgestalt/) [41,42]. We identified the genes whose expression was at least 20% different between the GC responder and non-responder BTM cell strains. User data and parameters:

Establishment of BTM Responder and Non-Responder Cell Strains
Three GC responder and non-responder BTM cell strains were established using the approach described in "Methods." Only confluent BTM cell cultures were used to mimic in vivo conditions [37,43]. Fig 1 demonstrates how BTM cell strains were categorized. One bovine eye was used for perfusion culture and DEX treatment, while the fellow eye was used for BTM cell establishment. According to the IOP response to DEX in the perfused eye, the BTM cell strain established from the fellow eye was defined as a responder or non-responder strain using our established criteria [29]. In the present study, we perfused 26 pairs of bovine eyes, and found that 7 pairs were responders with a responder rate of 36.8% which is very close to our published study [29]. DEX-induced OHT usually developed with 3-5 days. The mΔIOP of our responder cell strains selected for RNAseq ranged from 3 to 5.46 mmHg, while the mΔIOP of non-responder cell strains were no more than 1.53 mmHg ( Table 2).
We then treated the responder and non-responder BTM cells with 0.1% EtOH or 100nM DEX for 7 days and collected conditioned medium for WB. We found that all 3 responder cell strains showed an induction of fibronectin, a GC-inducible protein, while the 3 non-responder cell strains did not (Fig 2 and S1 Fig).

RNAseq Showed Differential DEX-Induced Gene Expression between BTM Responder and Non-Responder Cell Strains
The 6 BTM cell strains (3 responder and 3 non-responder) were treated with either EtOH or DEX for 7 days. RNA was extracted, analyzed for quality and quantity, and used for RNAseq library preparation. After expression quantitation, differential gene expression analysis was carried out using the differential expression groupings (DEG) strategy (Fig 3). Expression  The definition of individual DEG groups: DEG #1: Differentially expressed genes between DEX vs. EtOH treated responder cells.
RNAseq showed genes were differentially expressed in the presence of DEX between responder and non-responder groups   qPCR Validation We used qPCR to validate the expression of 3 of the most up-regulated and 3 of the most down-regulated genes in DEGs 3, 4 and 5 (total 18 genes, Table 3) identified by RNAseq. cDNA was prepared using the same RNA that was used for RNAseq. Since GAPDH showed significant changes to DEX treatment in the responder group (S2 Table), actin was used as an internal control. Our qPCR data closely matched RNAseq data (Fig 5) except for ALOX12, confirming the reliability of RNAseq technique.

Pathway Analysis
We used the WebGestalt tool to determine the biological pathways associated with DEGs 3, 4 and 5 (Fig 6). Our analysis identified 35 pathways which may play important roles in differential GC-responsiveness.

Discussion
We used the highly sensitive RNAseq technique to compare DEX-responder and nonresponder bovine TM cells. Different pools of genes were cross-compared, and a number of genes were found to be differentially expressed between responder and non-responder BTM cells. Pathway analyses showed that 35 pathways were closely associated with DEX responsiveness. Our results showed that GC-responder and non-responder TM cells react differently to GCs. Several studies explored DEX-induced gene expression changes in the TM using microarray techniques [31-36]. Although many genes were found to be differentially expressed upon DEX treatment, there was little consistency among those reports (Summarized in Table 4), which is very likely due to the use of TM cells of unknown GC-responsiveness. In contrast, our study is the first that has compared gene expression between TM cells isolated from eyes with known IOP and GC responsiveness.
Our data revealed differentially expressed genes that are involved in cell-adhesion, metabolism, extracellular matrix, and inflammatory response. Among these genes, we are particularly interested in Dickkopf 1 (DKK1) and K-Cadherin (CDH6). DKK1 is an inhibitor of the Wnt signaling pathway. We have previously reported that the Wnt pathway plays a role in regulating IOP in perfusion cultured human eyes and the mouse eye [43,44]. We also found that inhibition of the Wnt signaling pathway by DKK1 increased IOP [43]. One potential mechanism for this increase is through the stiffening of the trabecular meshwork [45,46]. Also, inhibition of the canonical Wnt signaling may promote ECM deposition [47]. In human osteoblasts, Ohnaka and colleagues found that DKK1 is up-regulated by GCs [48,49]. DKK1 is also increased in the extracellular matrix of DEX treated TM cells [45]. Therefore, DKK1 and the associated Wnt signaling, may play important roles in ocular GC responsiveness. SFRP1 is another Wnt pathway inhibitor, and we found that it is elevated in the glaucoma trabecular meshwork (GTM) and is able to induce OHT in mouse as well as human eyes [44].
In contrast to DKK1, the potential role of CDH6 in glaucoma pathogeneses is currently unclear. However, our preliminary studies suggest that CDH molecules may be involved in IOP regulation. CDH6 is expressed in the human trabecular meshwork (HTM), and our unpublished data show that CDH6 is able to inhibit SFRP1-induced OHT in mouse eyes. We believe that CDH6 and other cadherin molecules maintain TM homeostasis, and the disruption of these molecules contributes to OHT. Our hypothesis is supported by our findings that CDH6 was down-regulated by DEX treatment in both responder and non-responder TM cells, but the expression of this gene was more suppressed in responders (DEG 3). Besides Wnt and cell adhesion pathways, several well characterized pathways were also identified in this study. It is not surprising to find that the metabolic pathways of hexoses, polysaccharides and amino acids were among those pathways since they are known to be regulated by GCs. We also found cytokine and ECM related pathways are differentially regulated by GCs between responder and non-responders. Cytokines, especially Interleukin-6 (IL-6), have been extensively studied. IL-6 is induced by mechanical stress-induced TGFβ1 expression [50,51]. Liton and colleagues showed that IL-6 lowers outflow resistance in perfusion cultured porcine anterior segments, suggesting this cytokine may play a role in maintaining normal IOP, although Birke and colleagues found no hypotensive effects using a similar model [50,52]. We found that a number of inflammatory response-related molecules such as IL11/27RA, CXCL3 and CCL2/5, as well as IF16, IFI27 were down-regulated in the responder group (DEG 4, S2 Table). In contrast, pro-inflammatory proteins IL-6 and S100A12, were up-regulated by more than 1.5 or 39 fold, respectively, in the non-responder group (DEG 5, S3 Table). The difference in ILs and other inflammation associated molecules may contribute to the difference in IOP between the two groups. Another group of important molecules revealed by this study were molecules involved in ECM turnover. Numerous studies have shown that there is excessive ECM deposition in GIG and POAG TM cells and tissues. We found that both responder and non-responder groups had a down-regulation of MMP12, but this down-regulation in the responder group was much more than that in the non-responder group. In fact, MMP12 is the most down-regulated gene in DEG 3 (S1 Table). In addition to MMP12, up-regulation of TIMP1 and down-regulation of MMP3 were observed in response to DEX treatment in the responder group (S2 Table), but not in the non-responder group. MMPs and their inhibitors TIMPs work together to maintain TM ECM and IOP [53][54][55][56]. The dysregulation of MMP3, MMP12 and TIMP1 in the responder TM may provide further clues in explaining differential IOP responses. In addition to the pathways and genes that have known implications in the TM and POAG, the expression of many genes are altered by GCs in other tissues, but have not been reported in the POAG TM. For example, HMGA2, a member of the high mobility group AT-hook protein family that participates in DNA-protein interaction, plays a key role in chromatin architecture and gene regulation [57]. Overexpression of HMGA2 is associated with many types of tumors [57]. In POAG, loss of TM cells and fibrotic changes are observed, and TM cell senescence [58] may exacerbate these changes. The DEX induced up-regulation of HMGA2 may be a compensatory mechanism to antagonize TM cell senescence. Metallothionein 2A (MT2A), a metal binding enzyme, plays a role in anti-oxidant, anti-apoptosis, detoxification and antiinflammation [59]. It is also a GR-inducible gene in hepatic cells [60]. We found that MT2A is one of the most up-regulated genes in DEG#3 (Table 3), suggesting MT2A may be one of the factors that mediates GC responsiveness. Secreted protein acidic and rich in cysteine like protein 1 (SPARCL1) is a matrix protein whose level is associated with tumor metastasis and prognosis [61]. Naschberger et al. showed that SPARCL1 contributes to tumor endothelial cell quiescence [61]. In POAG TM, Rhee and colleagues found that SPARC plays a role in IOP regulation and TM pathology [62]. Although we found a DEX-induced down-regulation of SPARCL1 in responders, it may indicate a remodeling of the TM. Alternatively, the BTM may rely on different proteins compared to the HTM. Arachidonate 12-Lipoxygenase (ALOX12) metabolizes arachidonic acid as well as other lipids, and generates reactive oxygen species (ROS) [63]. Although the TM has a powerful system to handle oxidative stress throughout its lifespan [64], DEX-induced ALOX12 expression may increase the ROS burden of TM cells and therefore accelerate TM damage. The RNA component of mitochondrial RNA processing endoribonuclease (RMRP) gene is an untranslated gene. The transcript of the RMRP gene is a component of an RNA-dependent RNA polymerase complex consisting of RMRP RNA and TERT [65]. This complex is important for miRNA processing as well as cell and mitochondrial functions [65][66][67]. A DEX-induced increase in RMRP will likely to affect TM mitochondrial functions and inhibit a large number of genes [67]. The solute carrier family 2 member 5 (SLC2A5) gene encodes the fructose transporter GLUT5, and the aralkylamine N-acetyltransferase (AANAT) gene encodes an enzyme that plays a role in melatonin synthesis. Increased GLUT5 is found in tumor cells as a feature of their metabolism [68], while changes in AANAT is associated with depression [69]. Until now, most of the research of GIG are at cellular and molecular biology levels. The changes in SLC2A5 and AANAT suggest that the difference between GC responders and non-responders in their biochemistry is worthy of further investigation. Pentraxin 3 (PTX3) is a member of the pentraxin protein family. PTX3 is an inflammatory marker that has been found in immune cells and vascular cells [70]. However, many studies showed this protein may have a protective role on inflammation in the cardiovascular system [71,72]. In a clinical study, Lerzo and colleagues showed that DEX prophylaxis elevates PTX3 levels in pediatric patients receiving open heart surgeries, suggesting DEX induced PTX3 may contribute to decreased inflammation as well as improvement in prognosis [73]. Our data showed that PTX3 is one of the most upregulated genes in DEG5 (Table 3), indicating PTX3 may also protect the TM and prevent OHT in the non-responder eyes. The parathyroid hormone-like hormone (PTHLH) gene is a member of the parathyroid hormone family. PTHLH regulates endochondral bone development and epithelial mesenchymal interaction. Flöttmann et al. reported that duplication of PTHLH causes osteochondroplasia [74]. Since Borras and colleagues suggest that the pathological changes in the POAG TM resembles calcification [75], a down-regulation of PTHLH in the non-responder group may help to explain why their TM is less effected by DEX. The cellular retinoic acid binding protein 1 (CRABP) is involved in retinoic acid signaling. The role of retinoic acid in fibrosis is controversial [76]. Our findings that DEX selectively decreased CRABP1 in the non-responder group may suggest a profibrotic role of CRABP1 and retinoic acid in the TM. Further investigation of these genes previously not reported as being DEX responsive in the TM warrants further investigation. In another study using the in vivo bovine model, Danias and colleagues collected BTM tissues from cattle treated with or without topical prednisolone, and used bovine microarray to compare gene expression [77]. However, in that study, the authors reported a 100% responder rate and genes identified showed little overlap with our current data. We believe that the difference in cattle strains and GCs contribute to the discrepancy between the two studies. Also, the bovine genome continues to be updated. The genes detectable in microarrays vary based on how frequently the manufacturer updates their arrays. In contrast, RNAseq allows us to observe and map transcripts to the most up-to-date bovine assembly. Another advantage of RNAseq is its much wider dynamic range that enables the detection of transcripts expressed at very high and very low levels. In principle, RNAseq can detect the vast majority of RNA transcripts, including noncoding RNA.
In addition to the discovery of GIG related genes and pathways, our study provided useful technical information. For qPCR and microarray studies, a number of housekeeping genes are used as internal controls. However, the choice of housekeeping genes under various situations is often overlooked. We found that the GAPDH gene showed a 1.68 fold increase in response to DEX in the responder group but not in the non-responder group (S2 and S3 Tables). Since the RNAseq data are normalized using the FPKM method (see Methods) that does not reply on a single gene but the overall readings, we believe that our observation of differential expression of GAPDH is reliable. Therefore, we used actin as the internal control for our qPCR study instead of GAPDH since the expression of actin was not affected by DEX in either the responder or the non-responder groups (S2 Appendix). This selection may also be suitable for GC-related studied in HTM cells/tissues.
Although this study provided useful observations with respect to gene differential expression in GC responders and non-responders, there are still some unsolved questions. We were unable to detect significant DEX-induced myocilin (MYOC) expression, while we previously reported DEX-induced MYOC expression in conditioned medium from perfusion cultured bovine eyes [29]. Our unpublished data also showed that DEX rarely induces MYOC at protein or mRNA levels in BTM cell cultures, suggesting the expression MYOC is affected by adjacent tissues/environments in the bovine eye. Similarly, no DEX-induced MYOC expression in BTM cell cultures was reported in two other studies using BTM cells [43,78]. We previously showed that the induction of MYOC by DEX in HTM cells is a secondary, indirect response, requiring the DEX-induced synthesis of another protein [79]. We hypothesize that BTM cells may have a different MYOC induction pathway than HTM cells. Also, we were unable to compare the ratio of glucocorticoid receptor α (GRα) to GRβ due to the unavailability of bovine GRβ sequence. GRα is the functional GR receptor while the alternatively spliced GRβ acts as a dominant negative form isoform. The ratio of GRα to GRβ is a mechanism that may contribute to ocular GC-responsiveness [10,80]. Nevertheless, our RNAseq data suggest a potential alternative splicing site at the 3' end of GRα which may help us to identify GRβ (data not shown). Although we did not observe DEX-induced MYOC expression, we found CLAN formation in confluent cell cultures, which is a characteristic of TM cells. We also observed DEX-induced expression of caveolin 1 (CAV1), a GC-inducible gene. Aga and colleagues found that knockdown of CAV1 increases outflow facility in perfusion cultured human eyes [81]. In our study we found that CAV1 was up-regulated in DEG 4 (responder only genes), and this upregulation may contribute to decreased outflow facility and OHT.
In contrast to MYOC, we observed a clear difference in DEX-induced fibronectin expression using WB. Fibronectin is an important component of the ECM. In addition to mechanical support, it functions as a reservoir for many growth factors. For example, TGFβ2, a POAGassociated factor, binds to the latent TGF-β binding protein (LTBP). This protein complex anchors to fibronectin and other ECM molecules [82]. During tissue mechanical deformation or damage, there is a release of the active form of TGFβ2 which activates the TGFβ pathway [83]. Accumulation of excessive fibronectin is believed to increase outflow resistance in the TM and elevates IOP. Many studies showed that glaucoma-associated factors, including GCs, are able to induce fibronectin [10,38]. We found that the induction of fibronectin correlated well with BTM responsiveness and IOP elevation, and our findings suggest that fibronectin induction may be one of the contributing factors to GC-induced OHT. Interestingly, transcription factor binding analysis (http://www.sabiosciences.com/chipqpcrsearch.php?species_ id=0&factor=GR-beta&gene=fn1&nfactor=n&ninfo=n&ngene=n&B2=Search) shows that there are no GC response elements (GRE; GR binding sites) in the fibronectin gene, like the MYOC gene [79]. It is very likely that GC-induced fibronectin is a secondary response. Besides, there is a discrepancy between mRNA and protein levels of fibronectin. In contrast to DEXinduced fibronectin in conditioned medium collected from the responder group (Fig 2), the mRNA expression ratios of fibronectin did not show significant difference in either responders or non-responders (S2 Appendix). Since RNA and conditioned medium were collected simultaneously from the same cell cultures, we believe that this difference is likely due to post-translational processing. There are several possibilities: 1) microRNA (miRNA). DEX may induce miRNAs that affect fibronectin translation in non-responders since miRNA may repress translation without degrading mRNA; 2) protein cross-linking. It is well recognized that increased ECM protein cross-linking may slow down protein turnover. In this study, we found lysyl oxidase-like 2 (LOXL2), an enzyme that cross-links ECM proteins, is more elevated in the responder group (S1 Table); 3) protein degradation. As discussed previously, we found that MMP12 which degrades fibronectin, is significantly decreased in responders (S1 Table). Also, MMP3 is selectively decreased while TIMP1 is selectively increased in responders, but not in non-responders upon DEX treatment (S2 Table).
In conclusion, we developed an approach to establish bovine TM cell cultures with known GC responsiveness. Combined with the powerful RNAseq technique, we discovered a number of genes and pathways that may mediate differential GC responsiveness in the eye. Further studies are needed to determine the exact function of each gene/pathway in GC-induced OHT and GIG.