October 2011
Volume 52, Issue 11
Free
Biochemistry and Molecular Biology  |   October 2011
Quantitative Proteomics: TGFβ2 Signaling in Trabecular Meshwork Cells
Author Affiliations & Notes
  • Kathryn E. Bollinger
    From the Cole Eye Institute and
  • John S. Crabb
    From the Cole Eye Institute and
    Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio;
  • Xianglin Yuan
    From the Cole Eye Institute and
    Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio;
  • Tasneem Putliwala
    Department of Cell Biology and Anatomy, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas; and
  • Abbot F. Clark
    Department of Cell Biology and Anatomy, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas; and
  • John W. Crabb
    From the Cole Eye Institute and
    Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio;
    Departments of Ophthalmology and
    Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio.
  • Corresponding author: John W. Crabb, Cole Eye Institute, Cleveland Clinic Foundation (i-31), 9500 Euclid Avenue, Cleveland, OH 44195; crabbj@ccf.org
  • Footnotes
    2  Present affiliation: Department of Ophthalmology, Medical College of Georgia, Augusta, Georgia.
Investigative Ophthalmology & Visual Science October 2011, Vol.52, 8287-8294. doi:10.1167/iovs.11-8218
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Kathryn E. Bollinger, John S. Crabb, Xianglin Yuan, Tasneem Putliwala, Abbot F. Clark, John W. Crabb; Quantitative Proteomics: TGFβ2 Signaling in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(11):8287-8294. doi: 10.1167/iovs.11-8218.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: Transforming growth factor beta 2 (TGFβ2) is often elevated in the aqueous humor (AH) and trabecular meshwork (TM) of patients with primary open-angle glaucoma (POAG) and appears to contribute to POAG pathogenesis. To better understand TGFβ2 signaling in the eye, TGFβ2-induced proteomic changes were identified in cells cultured from the TM, a tissue involved in intraocular pressure (IOP) elevation in glaucoma.

Methods.: Primary cultures of human TM cells from four donors were treated with or without TGFβ2 (5 ng/mL) for 48 hours; then cellular protein was analyzed by liquid chromatography–mass spectrometry iTRAQ (isobaric tags for relative and absolute quantitation) technology.

Results.: A total of 853 proteins were quantified. TGFβ2 treatment significantly altered the abundance of 47 proteins, 40 of which have not previously been associated with TGFβ2 signaling in the eye. More than half the 30 elevated proteins support growing evidence that TGFβ2 induces extracellular matrix remodeling and abnormal cytoskeletal interactions in the TM. The levels of 17 proteins were reduced, including four cytoskeletal and six regulatory proteins. Both elevated and decreased regulatory proteins implicate TGFβ2-altered processes involving transcription, translation, and the glutamate/glutamine cycle. Altered levels of eight mitochondrial proteins support TGFβ2-induced mitochondrial dysfunction in the TM that in POAG could contribute to oxidative damage in the AH outflow pathway, TM senescence, and elevated IOP.

Conclusions.: The results expand the repertoire of proteins known to participate in TGFβ2 signaling, provide new molecular insight into POAG, and establish a quantitative proteomics database for the TM that includes candidate glaucoma biomarkers for future validation studies.

Primary open-angle glaucoma (POAG) is the most common form of the primary glaucomas and affects approximately 3 million Americans and more than 70 million people worldwide. 1 Visual loss in POAG results from damage to retinal ganglion cells and the optic nerve; advanced age and elevated intraocular pressure (IOP) are risk factors. 2,3 A significant proportion of POAG patients have what is termed normal-tension glaucoma in which IOP remains in the normal range and the cause of the neuropathy is unclear. 4 Nevertheless, IOP is still a major risk factor in normal-tension glaucoma because further lowering of IOP decreases disease progression. Despite the high prevalence of POAG and the identification of glaucoma susceptibility genes, 5 the molecular mechanisms of glaucoma are poorly understood. Elevated IOP in POAG appears related to pathologic changes in the aqueous humor (AH) outflow pathway that cause increased outflow resistance, particularly in the trabecular meshwork (TM). Abnormal accumulation of extracellular matrix (ECM), 6,7 abnormal protein expression, 8 and changes in cytoskeletal interactions 9,10 within the TM have been associated with increased AH outflow resistance and elevated IOP. Oxidative damage may also contribute to elevated IOP 11 and play a role in POAG. 12,13  
Transforming growth factor beta 2 (TGFβ2) is an immunosuppressive factor in normal human AH that helps maintain the immune privilege of the eye. 14,15 In addition to elevated levels of TGFβ2 in the AH of POAG patients, as shown in numerous studies, 16 18 TGFβ2 is elevated in glaucomatous TM tissues and cultured glaucomatous TM cells. 19 In vitro studies in the TM show that TGFβ2 can induce ECM remodeling, 20,21 inhibit cell proliferation, 22 induce senescence-like changes, 23 and alter the actin cytoskeleton. Elsewhere in the body, TGFβ2 mediates increased ECM deposition and has been implicated in fibrosis of the liver, 24 kidney, 25 and lung. 26 Gene expression studies have reported changes in TGFβ2-treated TM cells, 21,27,28 including increased levels of transcripts encoding ECM and cytoskeletal components. Organ culture studies in which the anterior segment of human and porcine eyes is perfused with TGFβ2 have reported increased IOP and ECM deposition in the AH drainage pathway. 21,29,30 More recently, adenoviral gene transfer of TGFβ2 in vivo to rodent eyes has led to reduced AH outflow and increased IOP. 31 This growing body of evidence implicates TGFβ2 in POAG pathology. 
To better understand the molecular consequences of TGFβ2 signaling in the anterior segment, we compared human TM cells with and without TGFβ2 treatment using global quantitative proteomics methods. Liquid chromatography–mass spectrometry (LC MS/MS) isobaric tags for relative and absolute quantitation (iTRAQ; Applied Biosystems, Foster City, CA) technology was used to quantify TGFβ2-induced protein changes in TM cells. Forty-seven significantly altered proteins were identified, 40 of which have not previously been associated with TGFβ2 signaling in the TM. The results provide new molecular insight into the consequences of TGFβ2 signaling in ocular hypertension and POAG. 
Methods
TM Cell Cultures
TM cells were isolated from postmortem human TM tissue explants derived from open-angle glaucoma and nonglaucomatous control donor eyes. Glaucoma status was indicated from donor medical histories. The average death to preservation time was 7.75 ± 3.3 hours. Eyes were stored at 4°C until the TM was dissected, generally within 24 to 36 hours. Primary cultures were established, and TM cell morphology and purity of these cultures were characterized as previously described. 22 Human TM cells were grown in Dulbecco's modified Eagle's medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 1% penicillin-streptomycin (HyClone), and 1% l-glutamine (Thermo-Scientific, Waltham, MA) to confluence in T-25 flasks or in six-well plates. Primary cultures of human TM cells from two POAG and two nonglaucomatous donors were treated with or without TGFβ2 (5 ng/mL) for 48 hours, yielding four TGFβ2-treated and four4 untreated TM cultures. 
Sample Preparation, iTRAQ Labeling, SCX Chromatography, Protein Identification and Quantitation, and Bioinformatic and Western Blot Analysis
Detailed methods are provided in the Supplementary Methods available online. Briefly, for proteomic analyses, proteins were extracted from TM cells, quantified by amino acid analysis, reduced, alkylated, and digested with trypsin. Tryptic peptides from TGFβ2-treated TM cells were labeled with iTRAQ tag 117 and mixed with an equal amount of tryptic peptides from the corresponding untreated cell sample labeled with iTRAQ tag 115. 32,33 Each peptide mixture was fractionated by strong cation exchange (SCX) chromatography, and fractions were collected for LC MS/MS. LC MS/MS was performed with a QTOF2 mass spectrometer equipped with a capillary column system (Cap LC; Waters Corporation, Milford, MA). 32,33 Protein identification used MASSLYNX 4.1 software (Waters), the Mascot search engine (Matrix Science), and the Swiss-Protein human sequence database. Protein quantitation from iTRAQ labeling was achieved with code written in the statistical program R. 32,33 Bioinformatic analyses were performed with The Protein ANalysis THrough Evolutionary Relationships (PANTHER) Classification System, pathway analysis (Ingenuity Pathways Analysis 8.5; Ingenuity Systems, Redwood City, CA), and the Swiss Protein database. Western blot analyses were performed on polyvinylidene difluoride membranes using established protocols and commercially available antibodies. 
Results
From four primary cultures of human TM cells treated or untreated with TGFβ2, a total of 853 proteins were identified and quantified with two or more peptides by LC MS/MS and iTRAQ quantification code written in R. The TM cell samples were from normal (n = 2) and glaucomatous (n = 2) donor eyes (Table 1). Quantitative results from each cell sample are presented in Supplementary Tables S1–S4, including protein ratios, SDs, P values, number of unique peptides quantified, percentage sequence coverage for each protein, and peptide false discovery rates. The data from all four TM samples were of comparable quality and appropriate for averaging based on consistently low peptide false discovery (average rates, 2.1% identity, 3.0% homology) and similar distributions of protein ratios. The average relative abundance of the 853 quantified proteins is presented in Supplementary Table S5, and the distribution of mean protein ratios for all proteins and those quantified in three or more TM samples (n = 450) are shown in Figure 1. The distributions in Figure 1 are slightly skewed, indicating that more proteins were increased in abundance than were decreased by TGFβ2 treatment. Criteria for determining whether a protein was elevated or decreased included the average adjusted protein ratio and P value, with only proteins quantified in three or more cell samples used for comparative purposes. Proteins exhibiting average protein ratios (adjusted by SEM) above or below the mean by at least 1 SD (Fig. 1) and P < 0.06 were considered of higher or lower abundance. 
Table 1.
 
Trabecular Meshwork Cell Samples
Table 1.
 
Trabecular Meshwork Cell Samples
Experiment Cell Culture Donor Age and Sex Cell Passage Protein Analyzed (μg)
1 NTM416-07 78/M 4 47
2 NTM496-05 82/F 3 54
3 GTM304-4 75/F 3 38
4 GTM477-02 85/F 4 67
Figure 1.
 
Distribution of protein ratios. The log2 mean distribution of protein ratios (TGFβ2-treated TM/untreated TM) are shown for (A) all 853 quantified proteins and (B) 450 proteins quantified in three or more cell samples. Median, mean, and SD values are indicated; protein ratios between 1 and 2 SD from the mean are shaded. The distribution of protein ratios is near normal for all proteins but with some spread above 1.0 (log2 mean ratio 0), suggesting the increased abundance of several proteins after TGFβ2 treatment.
Figure 1.
 
Distribution of protein ratios. The log2 mean distribution of protein ratios (TGFβ2-treated TM/untreated TM) are shown for (A) all 853 quantified proteins and (B) 450 proteins quantified in three or more cell samples. Median, mean, and SD values are indicated; protein ratios between 1 and 2 SD from the mean are shaded. The distribution of protein ratios is near normal for all proteins but with some spread above 1.0 (log2 mean ratio 0), suggesting the increased abundance of several proteins after TGFβ2 treatment.
Majority of the Determined Proteome Reflects That of Untreated TM Cells
Approximately 95% of the 853 quantified proteins and ∼90% of the 450 proteins quantified in three or more samples (Supplementary Table S5) were present in similar amounts in TGFβ2-treated and untreated TM cells. Accordingly, the overall determined proteome largely reflects that of untreated TM cells. The quantified proteins include ∼5% secreted, ∼46% cytoplasmic, ∼22% membrane and membrane-associated, ∼15% nuclear, and ∼12% mitochondrial proteins. Among the significant proteomic changes after TGFβ2 treatment, more secreted proteins were elevated (∼20%) than decreased (0%) and twice as many mitochondrial proteins were decreased (∼41%) than elevated (∼20%). 
TM Proteins Increased by TGFβ2
Thirty proteins were elevated ≥1 SD (P < 0.06) above the mean ratio after TGFβ2 treatment, including 11 proteins elevated ≥2 SD (Table 2). The majority of these proteins have not previously been associated with TGFβ2 signaling in the TM; however, more than 50% can be grouped by function into processes previously associated with TGFβ2 treatment, such as ECM remodeling and cytoskeletal interactions. For example, among the most elevated proteins were ECM-associated SPARC (secreted protein acidic and rich in cysteine), CTGF (connective tissue growth factor), cytoskeletal-associated myosin regulatory light chain 9, and caldesmon. Approximately 20% of the elevated proteins can be grouped into protein binding, folding, and chaperone processes, and another ∼26% can be grouped into regulatory processes involving transcription, translation, steroid binding, and mitochondrial metabolism. Proteins significantly increased in abundance by TGFβ2 treatment account for ∼7% of those quantified in three or more samples. 
Table 2.
 
Elevated Proteins in TGFβ2-Treated Human TM Cells
Table 2.
 
Elevated Proteins in TGFβ2-Treated Human TM Cells
SwissProt Accession Protein Subcellular Source Sample Frequency (Total = 4) Mean Protein Ratio SEM P
ECM Remodeling
P09486 SPARC† AC 3 6.70* 0.14 0.006
P29279 Connective tissue growth factor A 4 2.94* 0.18 0.009
P02461 Collagen α1 (III) A 4 2.89* 0.13 0.004
O00469 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2† C 4 2.58* 0.09 0.002
P07996 Thrombospondin-1† A 3 2.41* 0.15 0.030
Q16270 Insulin-like growth factor-binding protein 7 A 3 2.40* 0.17 0.036
O15460 Prolyl 4-hydroxylase subunit α2† C 4 2.11* 0.22 0.042
Q96CG8 Collagen triple helix repeat-containing protein 1 A 3 2.04* 0.14 0.036
P13674 Prolyl 4-hydroxylase subunit α1† C 4 1.53 0.14 0.057
P55145 Mesencephalic astrocyte-derived neurotrophic factor A 4 1.45 0.08 0.020
Cytoskeletal Interactions
P24844 Myosin regulatory light chain 9† B 4 2.12* 0.11 0.006
Q05682 Caldesmon B 4 2.06* 0.21 0.042
Q8WX93 Palladin B 4 1.70 0.06 0.003
Q9HBL0 Tensin-1 B 3 1.67 0.07 0.017
P37802 Transgelin-2 B 4 1.56 0.08 0.012
Q9Y2B0 Protein canopy homolog 2 C 3 1.45 0.03 0.008
Protein Folding and Chaperone Processes
P02511 α-Crystallin B B 4 3.37* 0.16 0.004
Q8NBS9 Thioredoxin domain-containing protein 5 C 4 1.66 0.06 0.003
Q9Y4L1 Hypoxia upregulated protein 1 C 4 1.49 0.09 0.020
Q96AY3 FK506-binding protein 10† C 4 1.48 0.05 0.005
P11021 78 kDa glucose-regulated protein C 4 1.43 0.06 0.008
Q15084 Protein disulfide-isomerase A6 C 4 1.41 0.10 0.041
Regulatory Processes
P50479 PDZ and LIM domain protein 4† B 4 1.68 0.14 0.036
Q14192 Four and a half LIM domain protein 2† BD 4 1.67 0.09 0.012
Q9P2E9 Ribosome-binding protein 1 C 4 1.65 0.07 0.006
O00264 Membrane-associated progesterone receptor component 1 C 4 1.53 0.14 0.059
P62851 40S ribosomal protein S25 B 4 1.50 0.12 0.045
O94925 Glutaminase, kidney isoform E 3 1.43 0.07 0.037
P24534 Elongation factor 1-β B 4 1.42 0.06 0.009
O15173 Membrane-associated progesterone receptor component 2 C 4 1.39 0.10 0.052
TM Proteins Decreased by TGFβ2
In response to TGFβ2 treatment, 17 proteins were decreased in abundance ≥1 SD (P < 0.06) below the mean ratio (Table 3); none of these proteins have previously been associated with TGFβ2 signaling in TM cells. CD9 antigen and mitochondrial superoxide dismutase 2 (SOD2) were the most significantly reduced proteins (decreased ∼64% and ∼46%, respectively). Four of the reduced proteins are associated with cytoskeletal interactions and six with a variety of regulatory processes, including transcription, translation, and the immune response. Notably, more than 40% of the decreased proteins were from the mitochondria and are involved in critical metabolic functions. Proteins significantly reduced in abundance accounted for ∼4% of those quantified in three or more samples. 
Table 3.
 
Decreased Proteins in TGFβ2-Treated Human TM Cells
Table 3.
 
Decreased Proteins in TGFβ2-Treated Human TM Cells
SwissProt Accession Protein Subcellular Source Sample Frequency (Total = 4) Mean Protein Ratio SEM P
Signaling/Regulation
P39023 60S ribosomal protein L3 B 4 0.69 0.11 0.044
Q71DI3 Histone H3.2 D 4 0.68 0.08 0.014
Q99536 Synaptic vesicle membrane protein VAT-1 homolog B 4 0.66 0.05 0.004
P01891 HLA class I histocompatibility antigen, A-68 α C 3 0.65 0.08 0.032
P04899 Guanine nucleotide-binding protein G(i), α2 B 4 0.59 0.10 0.012
P21926 CD9 antigen C 3 0.36* 0.24 0.051
Cytoskeletal Interactions
P07737 Profilin-1 B 4 0.70 0.12 0.059
Q9ULV4 Coronin-1C B 3 0.60 0.05 0.011
O00159 Myosin-1c BC 3 0.58 0.12 0.047
P68371 Tubulin β-2C B 3 0.57 0.05 0.007
Mitochondrial Metabolism
P45880 Voltage-dependent anion-selective channel protein 2 CE 4 0.73 0.05 0.011
P25705 ATP synthase subunit α CE 4 0.72 0.05 0.006
P55084 Trifunctional enzyme subunit β E 4 0.70 0.06 0.008
P24539 ATP synthase subunit β CE 4 0.68 0.11 0.037
P21796 Voltage-dependent anion-selective channel protein 1 CE 4 0.66 0.13 0.044
P00367 Glutamate dehydrogenase 1 E 4 0.60 0.10 0.012
P04179 Superoxide dismutase 2 [Mn] E 4 0.054* 0.06 0.002
Independent Evidence Supporting the ITRAQ Protein Quantitation
Western blot analysis was used to independently evaluate proteomic changes in the TM cell samples with and without TGFβ2 treatment. Densitometric results (Fig. 2) from immunoblots corroborated increased amounts of caldesmon and decreased amounts of SOD2 after TGFβ2 treatment and were in reasonable agreement with the iTRAQ data in both ratios and P values. Reports in the literature support TGFβ2-induced changes observed for at least six other proteins. Specifically, Western blot analyses published by others show that TGFβ2 treatment of human TM cells results in increased amounts of SPARC, 34 CTGF, 35 thrombospondin-1, 35 and α-crystallin B 36 and that CTGF treatment of human TM cells results in elevated levels of collagen α1 type III. 37 In addition, a previous proteomic study reported TGFβ2 induced increased amounts of transgelin-2, α-crystallin B, and possibly 78 kDa glucose-regulated protein in TM cells based on 2D gel spot staining intensity. 27  
Figure 2.
 
Western blot analysis. Western blot analysis was used to evaluate the relative amounts of proteins in TGFβ2-treated and untreated TM cells (n = 4 samples each). Average densitometry ratios (TGFβ2-treated/untreated) and P values (two-sided t-test) for caldesmon and SOD2 support the quantitative data shown in Tables 2 and 3. Previous protein analyses corroborate at least six additional TGFβ2-induced proteomic changes detected in this study (see Results).
Figure 2.
 
Western blot analysis. Western blot analysis was used to evaluate the relative amounts of proteins in TGFβ2-treated and untreated TM cells (n = 4 samples each). Average densitometry ratios (TGFβ2-treated/untreated) and P values (two-sided t-test) for caldesmon and SOD2 support the quantitative data shown in Tables 2 and 3. Previous protein analyses corroborate at least six additional TGFβ2-induced proteomic changes detected in this study (see Results).
Discussion
To better understand TGFβ2 signaling in the anterior segment, we quantified proteomic changes in four primary cultures of human TM cells after TGFβ2 treatment using LC MS/MS iTRAQ technology. The study was not designed to reveal differences between glaucomatous and nonglaucomatous cell cultures but rather to probe the impact of TGFβ2 signaling on TM cells. Although glaucomatous and normal TM cells may exhibit differences in TGFβ2 signaling yet to be identified, all TGFβ2-altered proteins in this study were derived from both TM donor populations and definitively demonstrate common consequences of TGFβ2 signaling in normal and glaucomatous TM cells. Most of the 853 quantified proteins (∼90%) were present in similar amounts in TGFβ2-treated and untreated cells, indicating that the determined proteome largely reflects that of untreated TM cells. TGFβ2 treatment significantly altered the abundance of 47 proteins; independent data from Western blot analysis and 2D-PAGE corroborated eight or more (∼17%) of these TGFβ2-induced proteomic changes. Bioinformatic analysis suggested that major biological processes associated with the quantified proteins (Fig. 3) include cellular metabolism (∼28% of the 853 proteins), signal transduction (∼8%), cell structure/motility (∼8%), immunity and defense (∼5%). and cell adhesion (2%). Biological analysis and interpretation (Ingenuity Pathway Analysis [IPA]; Ingenuity) of these proteins implicated gene expression and cellular assembly and organization as the highest scoring networks. 
Figure 3.
 
Biological functions of TM proteins. Functional classification of the 853 proteins quantified in human TM cells was performed with the PANTHER Classification System.
Figure 3.
 
Biological functions of TM proteins. Functional classification of the 853 proteins quantified in human TM cells was performed with the PANTHER Classification System.
Molecular Clues to POAG from TM Proteins Elevated by TGFβ2 Treatment
Thirty TM proteins were significantly increased in abundance after TGFβ2 treatment (Table 2), supporting the upregulated expression of these polypeptides. Analysis (IPA; Ingenuity) of the 30 elevated proteins suggested that TGFβ2 impacted two major networks, one involving amino acid metabolism, posttranslational modification, and small molecule biochemistry and the other involving gene expression, cellular compromise, and development. Seven of the elevated proteins (or their transcripts) were previously reported to be upregulated by TGFβ2; the other 23 elevated proteins provide an expanded profile of TGFβ2 signaling in TM cells. Though yet unconfirmed, calcification and mineralization of the TM have been hypothesized to contribute to elevated IOP in glaucoma, 38 and 30% of the elevated proteins bind either calcium or zinc, as noted in Table 2. Consistent with previous reports, 6,7,20,21 10 elevated proteins are involved in the structure and regulation of the ECM, and eight of these were increased more than twofold. SPARC, elevated more than sixfold, is a matricellular protein that binds multiple proteins and cell membranes and that has been hypothesized to contribute to elevated IOP in POAG in part because SPARC-null mice exhibit lower IOPs than wild-type controls. 39 The SPARC gene, highly expressed in nonglaucomatous TM, 40 has also been reported to be upregulated by mechanical stretching. 41 CTGF, a mediator of the effects of TGFβs on ECM remodeling, and thrombospondin-1, a major activator of latent TGFβs and elevated in the TM of some POAG patients, 42 were both found significantly elevated. Also elevated in this study were insulin-like growth factor-binding protein 7, which stimulates cell adhesion and mesencephalic astrocyte-derived neurotrophic factor (also known as Protein ARMET), which binds unfolded proteins and inhibits stress-induced cell death. In addition, levels of collagen α1 type III and several other proteins that modify collagen or collagen deposition in the ECM—namely procollagen-lysine 2-oxogluturate 5-dioxygenase 2, prolyl 4-hydroxylase α2 and α1, and collagen triple helix repeat-containing protein 1—were elevated. 
Previous studies suggest that the TM cytoskeleton is involved in regulating AH outflow and IOP, perhaps in part through the formation of cross-linked actin networks (CLANs). 9,10,43 CLANs are morphologically diverse structures in TM cells, appearing as tangles to geodesic dome-like, and observed in both normal and glaucomatous TM in vivo. 43 In this study, TGFβ2 induced apparent increased expression of the actin-binding proteins caldesmon, palladin, tensin-1, and transgelin-2, and cytoskeletal regulators like myosin regulatory light chain 9 and its binding partner protein canopy homolog 2. Altered expression of these proteins could promote CLAN formation and impact TM cell shape, motility, proliferation, contractile and tone properties, and cell-cell and cell-ECM interactions. 
Six proteins increased in abundance by TGFβ2 were associated with protein folding and chaperone activities, with α-crystallin B the most significantly elevated (>3-fold). Others in this group included thioredoxin domain-containing protein 5, hypoxia upregulated protein 1, FK506-binding protein 10 (also known as peptidyl-prolyl cis-trans isomerase FKBP10), 78 kDa glucose-regulated protein, and protein disulfide-isomerase A6. Overall, about twice as many proteins were elevated than decreased by TGFβ2 treatment, and the increased abundance of chaperones and folding proteins appear to be a stress response to metabolic changes, such as in protein synthesis and oxidation reduction. 
A variety of regulatory proteins were elevated ∼40% to 70% after TGFβ2 treatment. The possibility that TGFβ2 alters translational elongation is suggested by elevated levels of ribosome-binding protein 1, 40S ribosomal protein S25, and elongation factor 1β. TGFβ2-altered transcriptional regulation is also suggested by elevated levels of PDZ and LIM domain protein 4 and four and a half LIM domain protein 2, which bind zinc and a zinc finger binding protein. Also elevated were two steroid-binding proteins, membrane-associated progesterone receptor components 1 and 2, suggesting the possibility that TGFβ2 may play a role in steroid responsiveness in glucocorticoid-stimulated secondary glaucoma. 44 Mitochondrial glutaminase, an enzyme catalyzing the conversion of glutamine to glutamate, was elevated more than 40% after TGFβ2 treatment; notably, increased glutaminase activity in the rat retina has been associated with ocular hypertension. 45 Seven other mitochondrial proteins were altered by TGFβ2 treatment, as discussed. 
Molecular Clues to POAG from TM Proteins Decreased by TGFβ2 Treatment
The levels of 17 TM proteins were significantly reduced after TGFβ2 treatment (Table 3), implying downregulated expression, possible degradation, or both. Little is known about TGFβ2-downregulated processes in the TM. The highest scoring network from pathway analysis (IPA; Ingenuity) of the 17 decreased proteins concerns developmental disorders, renal and urological diseases, and gene expression. TGFβ2 lowered the abundance of four cytoskeletal components by ∼30% to 40%, including the actin-binding proteins profilin-1, coronin-1C, and myosin-1c and microtubule component tubulin β-2C. As noted, altered ratios of cytoskeletal proteins could promote the formation of CLANs and adversely impact TM cell properties and processes. 43 In addition to cytoskeletal proteins, a variety of regulatory proteins were decreased by ∼30% to 60%. CD9 antigen, a membrane protein involved in cell adhesion and motility, was the most significantly reduced (>60%). Decreased amounts of 60S ribosomal protein L3 and histone H3.2 implicate TGFβ2-altered translational elongation and transcription, respectively; all these processes are also potentially altered by other proteins elevated by TGFβ2 treatment, as discussed. Altered GTPase and ATPase activities are implied by TGFβ2-decreased levels of guanine nucleotide-binding protein Gi α2 and synaptic vesicle membrane protein VAT-1 homolog, respectively. Reduction of HLA class 1 histocompatability antigen A68α and CD9 antigen suggests TGFβ2-altered immune responses in TM cells, as has been proposed in POAG for retinal glial cells and the optic nerve head. 46  
Mitochondrial Dysfunction in the TM
In addition to elevated mitochondrial glutaminase, seven other mitochondrial proteins were significantly reduced by TGFβ2 treatment (Table 3). These decreased proteins serve important roles in energy production (e.g., ATP synthases), transmembrane transport (e.g., voltage-dependent anion channel proteins), and oxidation reduction (e.g., glutamate dehydrogenase 1 [GLUD1] and SOD2), and their decreased levels could severely impair cellular functions. Mitochondrial dysfunction in retinal ganglion cells has long been associated with glaucoma. 47,48 However, it is only recently that evidence suggests that elevated reactive oxygen species and impaired calcium regulation in TM cell mitochondria and TM mitochondrial DNA deletions may play a role in POAG. 49 51 The identification of eight mitochondrial proteins altered in abundance by TGFβ2 adds strong support to the hypothesis that mitochondrial dysfunction in the TM contributes to POAG. For example, TGFβ2 has been reported to induce senescence in the TM, 23 and elevated IOP has been reported to contribute to glutamate toxicity in the rat retina (by increasing glutaminase activity and reducing glutamate uptake). 45 We found mitochondrial glutaminase elevated by ∼43% and mitochondrial GLUD1 decreased by ∼40% in TM cells after TGFβ2 treatment. GLUD1 reduces the amount of glutamate by catalyzing its oxidation to 2-oxoglutarate. TGFβ2 may induce TM senescence and cell death through mitochondrial dysfunction that in part promotes glutamate imbalance. Elevated IOP has also been reported to decrease total SOD activity in the rat retina, 52 and we observed ∼46% reduction in mitochondrial SOD2 in TM cells after TGFβ2 treatment. Previous work has shown that ∼50% knockdown of mitochondrial SOD2 in mouse retinal pigment epithelium correlates with increased levels of oxidative protein modifications 53 and that TGFβ2 increases lipid peroxidation in human TM cells. 23 We and others (Bhattacharya SK, et al. IOVS. 2004;45:ARVO E-Abstract 4560) 54 have also observed increased levels of oxidative protein modifications in trabeculectomy tissues from POAG patients. Accordingly, the present data support the hypothesis that elevated IOP in POAG is due in part to TGFβ2-induced mitochondrial dysfunction in the TM, with concomitant oxidative damage in the AH outflow pathway. 
Comparison with Other Studies of TGFβ2-Induced Changes in TM Protein and Gene Expression
This is the most extensive quantitative proteomic analysis to date of changes in human TM cells in response to TGFβ2. The only other global proteomic study of TGFβ2-treated TM cells used peptide mass fingerprinting for protein identification and 2D gel staining intensity for protein quantification. 27 Of the 56 proteins reported in the previous study, we detected 42 and obtained reliable quantitation (i.e., proteins found in three or more TM samples) for 12 of the 24 proteins reported to exhibit a significant change in 2D gel spot intensity. 27 For these 12 proteins, two to three exhibited changes in agreement with the present results, namely increased amounts of transgelin-2, α-crystallin B, and possibly 78 kDa glucose-regulated protein (which was reported as 1 of 2 proteins in a gel spot). 27 Comparison of these proteomic studies is complicated by the different methods used, including the analysis of pooled rather than individual donor TM samples and a lower concentration of TGFβ2 for treating TM cells in the earlier study. Comparison of the present results with gene expression studies of TGFβ2-treated TM cells 21,27,28 reveals limited correlation, however; only ∼20% correlation generally exists between transcript and cognate protein levels in mammals. 55 From microarray studies, more than 20 transcripts have been reported to be differentially expressed by TGFβ2 in human TM cells. 21,27,28 Significant proteomic changes in this study are consistent with reported TGFβ2 upregulation of transcripts for thrombospondin 1, 21,27 SPARC, 23 and prolyl 4 hydroxylase subunit alpha 2. 28 Proteases and protease inhibitors function in ECM remodeling in normal TM, 56 and TGFβ2 has been reported to upregulate the expression of the inactive proform of matrix metalloproteinase-2 57 and plasminogen activator inhibitor-1 in TM cells. 21,35,57 Surprisingly, few proteases or protease inhibitors were among the 853 proteins quantified in this study, perhaps because they were below detection limits. Nevertheless, we found almost twice as many proteins increased rather than decreased in abundance, suggesting that TGFβ2 signaling may inhibit proteolytic processes in the TM. 57 Several proteins previously reported to modify or to be associated with TGFβ2 signaling in the TM or to be overexpressed in POAG TM, including bone morphogenetic protein family members gremlin, Smad 7, and tissue transglutaminase and the lysyl oxidase family of enzymes, were not detected in this study. 28,35,58 60 Again, these proteins may be below the detection limits of this study. In addition, no known glaucoma susceptibility gene products were detected. We did detect two proteins reportedly upregulated in TM by TGFβ2, namely senescence-associated-β-galactosidase 23 and fibronectin, 21,35 but the abundance of these proteins was not significantly different between TGFβ2-treated and untreated cells. Other TGFβ2-regulated proteins certainly exist in the TM, and some may be among those (Supplementary Table S5) that narrowly miss the criteria used for a significant change in this study. 
The mechanism or mechanisms responsible for elevated expression of TGFβ2 in glaucoma patients are unknown. TGFβ2 in the aqueous humor could be made and secreted from one or more tissues surrounding the anterior segment, including the ciliary epithelium, iris, lens epithelium, corneal endothelium, and TM. Elevated levels of TGFβ2 in glaucomatous TM tissues could be derived in part from the aqueous humor and bound to the TM extracellular matrix. 19 However, glaucomatous TM cells express a latent isoform of TGFβ2 of higher molecular weight that must be activated to become biologically active. 19 How latent TGFβ2 is activated in TM cells remains unclear but may involve thrombospondin-1, 42 which was found elevated in this study. 
In summary, the present results add strong support to evidence that TGFβ2 can induce ECM remodeling and alter cytoskeletal interactions in the TM. In addition, the results implicate TGFβ2-altered regulation in processes such as transcription, translation, steroid metabolism, and the glutamate/glutamine cycle. Finally, the data provide new evidence that TGF-β2-induces mitochondrial dysfunction in the TM that could contribute to oxidative damage in the AH outflow pathway, TM senescence, and ocular hypertension in POAG. Overall, the results expand the repertoire of proteins known to participate in TGFβ2 signaling and establish a quantitative proteomic database for the TM that includes candidate glaucoma biomarkers for future validation studies. 
Supplementary Materials
Text s1, PDF - Text s1, PDF 
Footnotes
 Supported in part by National Institutes of Health Grants EY018147, EY14239, and EY15638; a Research to Prevent Blindness Challenge grant; American Health Assistance Foundation Grant 34-0714585 (KEB); an RPB Senior Investigator Award (JWC); a Steinbach Award (JWC); and The Cleveland Clinic Foundation.
Footnotes
 Disclosure: K.E. Bollinger, None; J.S. Crabb, None; X. Yuan, None; T. Putliwala, None; A.F. Clark, None; J.W. Crabb, Alcon (C), Allergan (C)
The authors thank Phillip Howe, Joe Hollyfield, Edward Rockwood, Andrew Schachat, and Elias Traboulsi for valuable discussions. 
References
Quigley HA Broman AT . The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol. 2006;90:262–267. [CrossRef] [PubMed]
Sommer A Tielsch JM Katz J . Relationship between intraocular pressure and primary open angle glaucoma among white and black Americans: the Baltimore Eye Survey. Arch Ophthalmol. 1991;109:1090–1095. [CrossRef] [PubMed]
Leske MC Wu SY Honkanen R . Nine-year incidence of open-angle glaucoma in the Barbados Eye Studies. Ophthalmology. 2007;114:1058–1064. [CrossRef] [PubMed]
Nemesure B Honkanen R Hennis A Wu SY Leske MC . Incident open-angle glaucoma and intraocular pressure. Ophthalmology. 2007;114:1810–1815. [CrossRef] [PubMed]
Wiggs JL . Genetic etiologies of glaucoma. Arch Ophthalmol. 2007;125:30–37. [CrossRef] [PubMed]
Lutjen-Drecoll E Shimizu T Rohrbach M Rohen JW . Quantitative analysis of ‘plaque material’ in the inner- and outer wall of Schlemm's canal in normal- and glaucomatous eyes. Exp Eye Res. 1986;42:443–455. [CrossRef] [PubMed]
Acott TS Kingsley PD Samples JR Van Buskirk EM . Human trabecular meshwork organ culture: morphology and glycosaminoglycan synthesis. Invest Ophthalmol Vis Sci. 1988;29:90–100. [PubMed]
Bhattacharya SK Rockwood EJ Smith SD . Proteomics reveal cochlin deposits associated with glaucomatous trabecular meshwork. J Biol Chem. 2005;280:6080–6084. [CrossRef] [PubMed]
Clark A Wilson K McCartney M Miggans S Kunkle M Howe W . Glucocorticoid-induced formation of cross-linked actin networks in cultured human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 1994;35:281–294. [PubMed]
Read AT Chan DW Ethier CR . Actin structure in the outflow tract of normal and glaucomatous eyes. Exp Eye Res. 2007;84:214–226. [CrossRef] [PubMed]
Kahn M Giblin F Epstein D . Glutathione in calf trabecular meshwork and its relation to aqueous humor outflow facility. Invest Ophthalmol Vis Sci. 1983;24:1283–1287. [PubMed]
Tezel G . Oxidative stress in glaucomatous neurodegeneration: mechanisms and consequences. Prog Retin Eye Res. 2006;25:490–513. [CrossRef] [PubMed]
Sacca SC Izzotti A Rossi P Traverso C . Glaucomatous outflow pathway and oxidative stress. Exp Eye Res. 2007;84:389–399. [CrossRef] [PubMed]
Cousins SW McCabe MM Danielpour D Streilein JW . Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor. Invest Ophthalmol Vis Sci. 1991;32:2201–2211. [PubMed]
Wilbanks GA Mammolenti M Streilein JW . Studies on the induction of anterior chamber-associated immune deviation (ACAID), III: induction of ACAID depends upon intraocular transforming growth factor-beta. Eur J Immunol. 1992;22:165–173. [CrossRef] [PubMed]
Tripathi R Li J Chan W Tripathi B . Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2. Exp Eye Res. 1994;59:723–727. [CrossRef] [PubMed]
Inatani M Tanihara H Katsuta H Honjo M Kido N Honda Y . Transforming growth factor-beta 2 levels in aqueous humor of glaucomatous eyes. Graefes Arch Clin Exp Ophthalmol. 2001;239:109–113. [CrossRef] [PubMed]
Picht G Welge-Luessen U Grehn F Lutjen-Drecoll E . Transforming growth factor beta 2 levels in the aqueous humor in different types of glaucoma and the relation to filtering bleb development. Graefes Arch Clin Exp Ophthalmol. 2001;239:199–207. [CrossRef] [PubMed]
Tovar-Vidalesa T Clark AF Wordinger RJ . Transforming growth factor-beta2 utilizes the canonical Smad-signaling pathway to regulate tissue transglutaminase expression in human trabecular meshwork cells. Exp Eye Res. 2011 June 24. [Epub ahead of print].
Fuchshofer R Birke M Welge-Lussen U Kook D Lutjen-Drecoll E . Transforming growth factor-beta 2 modulated extracellular matrix component expression in cultured human optic nerve head astrocytes. Invest Ophthalmol Vis Sci. 2005;46:568–578. [CrossRef] [PubMed]
Fleenor DL Shepard AR Hellberg PE Jacobson N Pang IH Clark AF . TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure. Invest Ophthalmol Vis Sci. 2006;47:226–234. [CrossRef] [PubMed]
Wordinger R Clark A Agarwal R . Cultured human trabecular meshwork cells express functional growth factor receptors. Invest Ophthalmol Vis Sci. 1998;39:1575–1589. [PubMed]
Yu AL Birke K Moriniere J Welge-Lussen U . TGF-beta2 induces senescence-associated changes in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2010;51:5718–5723. [CrossRef] [PubMed]
Gressner AM Weiskirchen R . Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med. 2006;10:76–99. [CrossRef] [PubMed]
Liu Y . Renal fibrosis: new insights into the pathogenesis and therapeutics. Kidney Int. 2006;69:213–217. [CrossRef] [PubMed]
Willis BC Borok Z . TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol. 2007;293:L525–L534. [CrossRef] [PubMed]
Zhao X Ramsey KE Stephan DA Russell P . Gene and protein expression changes in human trabecular meshwork cells treated with transforming growth factor-beta. Invest Ophthalmol Vis Sci. 2004;45:4023–4034. [CrossRef] [PubMed]
Fuchshofer R Stephan D Russell P Tamm E . Gene expression profiling of TGFbeta2- and/or BMP7-treated trabecular meshwork cells: Identification of Smad7 as a critical inhibitor of TGF-beta2 signaling. Exp Eye Res. 2009;88:1020–1032. [CrossRef] [PubMed]
Gottanka J Chan D Eichhorn M Lutjen-Drecoll E Ethier CR . Effects of TGF-beta2 in perfused human eyes. Invest Ophthalmol Vis Sci. 2004;45:153–158. [CrossRef] [PubMed]
Bachmann B Birke M Kook D Eichhorn M Lutjen-Drecoll E . Ultrastructural and biochemical evaluation of the porcine anterior chamber perfusion model. Invest Ophthalmol Vis Sci. 2006;47:2011–2020. [CrossRef] [PubMed]
Shepard AR Millar JC Pang IH Jacobson N Wang WH Clark AF . Adenoviral gene transfer of active human transforming growth factor-beta 2 elevates intraocular pressure and reduces outflow facility in rodent eyes. Invest Ophthalmol Vis Sci. 2010;51:2067–2076. [CrossRef] [PubMed]
Crabb JW Yuan X Dvoriantchikova G Ivanov D Crabb JS Shestopalov VI . Preliminary quantitative proteomic characterization of glaucomatous rat retinal ganglion cells. Exp Eye Res. 2010;91:107–110. [CrossRef] [PubMed]
Yuan X Gu X Crabb JS . Quantitative proteomics: comparison of the macular Bruch membrane/choroid complex from age-related macular degeneration and normal eyes. Mol Cell Proteomics. 2010;9:1031–1046. [CrossRef] [PubMed]
Kang MH Oh DJ Rhee DJ . Effect of hevin deletion in mice and characterization in trabecular meshwork. Invest Ophthalmol Vis Sci. 2011;52:2187–2193. [CrossRef] [PubMed]
Fuchshofer R Yu A Welge-Lüssen U Tamm E . Bone morphogenetic protein-7 is an antagonist of transforming growth factor-beta2 in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2007;48:715–726. [CrossRef] [PubMed]
Welge-Lussen U May CA Eichhorn M Bloemendal H Lutjen-Drecoll E . AlphaB-crystallin in the trabecular meshwork is inducible by transforming growth factor-beta. Invest Ophthalmol Vis Sci. 1999;40:2235–2241. [PubMed]
Junglas B Yu AH Welge-Lussen U Tamm ER Fuchshofer R . Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells. Exp Eye Res. 2009;88:1065–1075. [CrossRef] [PubMed]
Borras T Comes N . Evidence for a calcification process in the trabecular meshwork. Exp Eye Res. 2009;88:738–746. [CrossRef] [PubMed]
Haddadin RI Oh DJ Kang MH . SPARC-null mice exhibit lower intraocular pressures. Invest Ophthalmol Vis Sci. 2009;50:3771–3777. [CrossRef] [PubMed]
Tomarev SI Wistow G Raymond V Dubois S Malyukova I . Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis. Invest Ophthalmol Vis Sci. 2003;44:2588–2596. [CrossRef] [PubMed]
Vittal V Rose A Gregory KE Kelley MJ Acott TS . Changes in gene expression by trabecular meshwork cells in response to mechanical stretching. Invest Ophthalmol Vis Sci. 2005;46:2857–2868. [CrossRef] [PubMed]
Flugel-Koch C Ohlmann A Fuchshofer R Welge-Lussen U Tamm ER . Thrombospondin-1 in the trabecular meshwork: localization in normal and glaucomatous eyes, and induction by TGF-beta1 and dexamethasone in vitro. Exp Eye Res. 2004;79:649–663. [CrossRef] [PubMed]
Hoare MJ Grierson I Brotchie D Pollock N Cracknell K Clark AF . Cross-linked actin networks (CLANs) in the trabecular meshwork of the normal and glaucomatous human eye in situ. Invest Ophthalmol Vis Sci. 2009;50:1255–1263. [CrossRef] [PubMed]
Clark AF Wordinger RJ . The role of steroids in outflow resistance. Exp Eye Res. 2009;88:752–759. [CrossRef] [PubMed]
Moreno MC Sande P Marcos HA . Effect of glaucoma on the retinal glutamate/glutamine cycle activity. FASEB J. 2005;19:1161–1162. [PubMed]
Tezel G . The role of glia, mitochondria, and the immune system in glaucoma. Invest Ophthalmol Vis Sci. 2009;50:1001–1012. [CrossRef] [PubMed]
Kong G Van Bergen N Trounce I Crowston J . Mitochondrial dysfunction and glaucoma. J Glaucoma. 2009;18:93–100. [CrossRef] [PubMed]
Osborne N . Mitochondria: their role in ganglion cell death and survival in primary open angle glaucoma. Exp Eye Res. 2010;90:750–757. [CrossRef] [PubMed]
He Y Leung KW Zhang YH . Mitochondrial complex I defect induces ROS release and degeneration in trabecular meshwork cells of POAG patients: protection by antioxidants. Invest Ophthalmol Vis Sci. 2008;49:1447–1458. [CrossRef] [PubMed]
He Y Ge J Tombran-Tink J . Mitochondrial defects and dysfunction in calcium regulation in glaucomatous trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2008;49:4912–4922. [CrossRef] [PubMed]
Izzotti A Longobardi M Cartiglia C Sacca SC . Mitochondrial damage in the trabecular meshwork occurs only in primary open-angle glaucoma and in pseudoexfoliative glaucoma. PLoS ONE. 2011;6:e14567. [CrossRef] [PubMed]
Moreno MC Campanelli J Sande P . Retinal oxidative stress induced by high intraocular pressure. Free Radic Biol Med. 2004;37:803–812. [CrossRef] [PubMed]
Justilien V Pang J Renganathan K . SOD2 knockdown mouse model of early AMD. Invest Ophthalmol Vis Sci. 2007;48:4407–4420. [CrossRef] [PubMed]
Govindarajan B Laird J Salomon RG Bhattacharya SK . Isolevuglandin-modified proteins, including elevated levels of inactive calpain-1, accumulate in glaucomatous trabecular meshwork. Biochemistry. 2008;47:817–825. [CrossRef] [PubMed]
Tian Q Stepaniants SB Mao M . Integrated genomic and proteomic analyses of gene expression in mammalian cells. Mol Cell Proteomics. 2004;3:960–969. [CrossRef] [PubMed]
Keller KE Aga M Bradley JM Kelley MJ Acott TS . Extracellular matrix turnover and outflow resistance. Exp Eye Res. 2009;88:676–682. [CrossRef] [PubMed]
Fuchshofer R Welge-Lussen U Lutjen-Drecoll E . The effect of TGF-beta2 on human trabecular meshwork extracellular proteolytic system. Exp Eye Res. 2003;77:757–765. [CrossRef] [PubMed]
Wordinger RJ Fleenor DL Hellberg PE . Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma. Invest Ophthalmol Vis Sci. 2007;48:1191–1200. [CrossRef] [PubMed]
Tovar-Vidales T Roque R Clark AF Wordinger RJ . Tissue transglutaminase expression and activity in normal and glaucomatous human trabecular meshwork cells and tissues. Invest Ophthalmol Vis Sci. 2008;49:622–628. [CrossRef] [PubMed]
Sethi A Mao W Wordinger RJ Clark AF . Transforming growth factor beta induces extracellular matrix protein crosslinking lysyl oxidase (LOX) genes in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2011;52:5240–5250. [CrossRef] [PubMed]
Figure 1.
 
Distribution of protein ratios. The log2 mean distribution of protein ratios (TGFβ2-treated TM/untreated TM) are shown for (A) all 853 quantified proteins and (B) 450 proteins quantified in three or more cell samples. Median, mean, and SD values are indicated; protein ratios between 1 and 2 SD from the mean are shaded. The distribution of protein ratios is near normal for all proteins but with some spread above 1.0 (log2 mean ratio 0), suggesting the increased abundance of several proteins after TGFβ2 treatment.
Figure 1.
 
Distribution of protein ratios. The log2 mean distribution of protein ratios (TGFβ2-treated TM/untreated TM) are shown for (A) all 853 quantified proteins and (B) 450 proteins quantified in three or more cell samples. Median, mean, and SD values are indicated; protein ratios between 1 and 2 SD from the mean are shaded. The distribution of protein ratios is near normal for all proteins but with some spread above 1.0 (log2 mean ratio 0), suggesting the increased abundance of several proteins after TGFβ2 treatment.
Figure 2.
 
Western blot analysis. Western blot analysis was used to evaluate the relative amounts of proteins in TGFβ2-treated and untreated TM cells (n = 4 samples each). Average densitometry ratios (TGFβ2-treated/untreated) and P values (two-sided t-test) for caldesmon and SOD2 support the quantitative data shown in Tables 2 and 3. Previous protein analyses corroborate at least six additional TGFβ2-induced proteomic changes detected in this study (see Results).
Figure 2.
 
Western blot analysis. Western blot analysis was used to evaluate the relative amounts of proteins in TGFβ2-treated and untreated TM cells (n = 4 samples each). Average densitometry ratios (TGFβ2-treated/untreated) and P values (two-sided t-test) for caldesmon and SOD2 support the quantitative data shown in Tables 2 and 3. Previous protein analyses corroborate at least six additional TGFβ2-induced proteomic changes detected in this study (see Results).
Figure 3.
 
Biological functions of TM proteins. Functional classification of the 853 proteins quantified in human TM cells was performed with the PANTHER Classification System.
Figure 3.
 
Biological functions of TM proteins. Functional classification of the 853 proteins quantified in human TM cells was performed with the PANTHER Classification System.
Table 1.
 
Trabecular Meshwork Cell Samples
Table 1.
 
Trabecular Meshwork Cell Samples
Experiment Cell Culture Donor Age and Sex Cell Passage Protein Analyzed (μg)
1 NTM416-07 78/M 4 47
2 NTM496-05 82/F 3 54
3 GTM304-4 75/F 3 38
4 GTM477-02 85/F 4 67
Table 2.
 
Elevated Proteins in TGFβ2-Treated Human TM Cells
Table 2.
 
Elevated Proteins in TGFβ2-Treated Human TM Cells
SwissProt Accession Protein Subcellular Source Sample Frequency (Total = 4) Mean Protein Ratio SEM P
ECM Remodeling
P09486 SPARC† AC 3 6.70* 0.14 0.006
P29279 Connective tissue growth factor A 4 2.94* 0.18 0.009
P02461 Collagen α1 (III) A 4 2.89* 0.13 0.004
O00469 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2† C 4 2.58* 0.09 0.002
P07996 Thrombospondin-1† A 3 2.41* 0.15 0.030
Q16270 Insulin-like growth factor-binding protein 7 A 3 2.40* 0.17 0.036
O15460 Prolyl 4-hydroxylase subunit α2† C 4 2.11* 0.22 0.042
Q96CG8 Collagen triple helix repeat-containing protein 1 A 3 2.04* 0.14 0.036
P13674 Prolyl 4-hydroxylase subunit α1† C 4 1.53 0.14 0.057
P55145 Mesencephalic astrocyte-derived neurotrophic factor A 4 1.45 0.08 0.020
Cytoskeletal Interactions
P24844 Myosin regulatory light chain 9† B 4 2.12* 0.11 0.006
Q05682 Caldesmon B 4 2.06* 0.21 0.042
Q8WX93 Palladin B 4 1.70 0.06 0.003
Q9HBL0 Tensin-1 B 3 1.67 0.07 0.017
P37802 Transgelin-2 B 4 1.56 0.08 0.012
Q9Y2B0 Protein canopy homolog 2 C 3 1.45 0.03 0.008
Protein Folding and Chaperone Processes
P02511 α-Crystallin B B 4 3.37* 0.16 0.004
Q8NBS9 Thioredoxin domain-containing protein 5 C 4 1.66 0.06 0.003
Q9Y4L1 Hypoxia upregulated protein 1 C 4 1.49 0.09 0.020
Q96AY3 FK506-binding protein 10† C 4 1.48 0.05 0.005
P11021 78 kDa glucose-regulated protein C 4 1.43 0.06 0.008
Q15084 Protein disulfide-isomerase A6 C 4 1.41 0.10 0.041
Regulatory Processes
P50479 PDZ and LIM domain protein 4† B 4 1.68 0.14 0.036
Q14192 Four and a half LIM domain protein 2† BD 4 1.67 0.09 0.012
Q9P2E9 Ribosome-binding protein 1 C 4 1.65 0.07 0.006
O00264 Membrane-associated progesterone receptor component 1 C 4 1.53 0.14 0.059
P62851 40S ribosomal protein S25 B 4 1.50 0.12 0.045
O94925 Glutaminase, kidney isoform E 3 1.43 0.07 0.037
P24534 Elongation factor 1-β B 4 1.42 0.06 0.009
O15173 Membrane-associated progesterone receptor component 2 C 4 1.39 0.10 0.052
Table 3.
 
Decreased Proteins in TGFβ2-Treated Human TM Cells
Table 3.
 
Decreased Proteins in TGFβ2-Treated Human TM Cells
SwissProt Accession Protein Subcellular Source Sample Frequency (Total = 4) Mean Protein Ratio SEM P
Signaling/Regulation
P39023 60S ribosomal protein L3 B 4 0.69 0.11 0.044
Q71DI3 Histone H3.2 D 4 0.68 0.08 0.014
Q99536 Synaptic vesicle membrane protein VAT-1 homolog B 4 0.66 0.05 0.004
P01891 HLA class I histocompatibility antigen, A-68 α C 3 0.65 0.08 0.032
P04899 Guanine nucleotide-binding protein G(i), α2 B 4 0.59 0.10 0.012
P21926 CD9 antigen C 3 0.36* 0.24 0.051
Cytoskeletal Interactions
P07737 Profilin-1 B 4 0.70 0.12 0.059
Q9ULV4 Coronin-1C B 3 0.60 0.05 0.011
O00159 Myosin-1c BC 3 0.58 0.12 0.047
P68371 Tubulin β-2C B 3 0.57 0.05 0.007
Mitochondrial Metabolism
P45880 Voltage-dependent anion-selective channel protein 2 CE 4 0.73 0.05 0.011
P25705 ATP synthase subunit α CE 4 0.72 0.05 0.006
P55084 Trifunctional enzyme subunit β E 4 0.70 0.06 0.008
P24539 ATP synthase subunit β CE 4 0.68 0.11 0.037
P21796 Voltage-dependent anion-selective channel protein 1 CE 4 0.66 0.13 0.044
P00367 Glutamate dehydrogenase 1 E 4 0.60 0.10 0.012
P04179 Superoxide dismutase 2 [Mn] E 4 0.054* 0.06 0.002
Text s1, PDF
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×