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(Investigative Ophthalmology and Visual Science. 2004;45:2168-2176.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.03-1218
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Downstream Effects of ROCK Signaling in Cultured Human Corneal Stromal Cells: Microarray Analysis of Gene Expression

Stephen A. K. Harvey, Susan C. Anderson, and Nirmala SundarRaj

From the Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania.


   Abstract
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 Abstract
Methods and Materials
 Results and Discussion
 References
 
PURPOSE. Rho-associated coiled-coil-containing protein kinase (ROCK) is a downstream target of Rho GTPase signaling and regulates the assembly of stress fibers. Previous reports indicate that Rho/ROCK signaling is involved in the regulation of several cellular processes, some of which may be cell-type specific and are probably critical to corneal stromal cell activation. The present study identified ROCK-regulated gene expression in corneal stromal cells.

METHODS. Corneal stromal cells derived from eyes of three different donors were cultured to yield the following designated phenotypes: baseline fibroblasts (DMEM with 10% serum), activated fibroblasts (10% serum+bFGF+heparin), and myofibroblasts (1% serum+TGF-ß1). Cells were exposed to the ROCK inhibitor Y-27632 or vehicle for 12 hours, and transcript levels altered by ROCK inhibition were identified with oligonucleotide microarrays (GeneChips; Affymetrix, Santa Clara, CA).

RESULTS. In these phenotypes, Y-27632 caused marked (twofold or more) increases or decreases in 14/4, 12/3, and 15/10 transcripts. In both fibroblast groups Y-27632-treatment increased expression of endothelin receptors and of parathyroid hormone-like hormone. The upregulation of {alpha}-smooth muscle actin in myofibroblasts was attenuated by Y-27632. Combining data from all groups identified ROCK-supported (Y-27632 inhibitable) expression of 10 transcripts, including ribonucleotide reductase M2, the cyclin B1-CDC2-CKS2 system, and four mitotic spindle-associated proteins.

CONCLUSIONS. ROCK inhibition causes broad inhibition of DNA synthesis and mitosis and causes changes that are different between (bFGF-activated) fibroblasts and (TGF-ß1–induced) myofibroblasts. Thus, Rho/ROCK signaling regulates both common and distinct downstream events in corneal stromal cells activated (differentiated) to fibroblast or myofibroblast phenotype.

Rho, a homologue of small GTPase Ras, is involved in many actin-associated cellular processes leading to nuclear signaling (for reviews, see Refs. 1 2 3 4 5 6 7 8 9 10 11 ). The recent identification and characterization of several Rho-binding proteins illuminates the biochemical mechanisms of specific downstream effects. One of the Rho targets is Rho-associated coiled-coil-containing protein kinase (ROCK), a kinase that is involved in mediating the assembly of focal adhesions and stress fibers. Two isoforms of (ROCK-I/ROKß and ROCK-II/ROK{alpha})12 13 14 15 have been identified. ROCK phosphorylates a myosin phosphatase and thereby suppresses its phosphatase activity,16 17 which leads to increases in the levels of phosphorylated myosin and induction of actomyosin-based contraction. ROCK has been shown also to phosphorylate and thereby activate LIM kinase.18 19 LIM kinase can phosphorylate and inactivate an actin depolymerizing protein, switching off actin filament disassembly. We have reported that increased expression of ROCK is associated with limbal epithelial transition to corneal epithelium.13 ROCK also influences the assembly of E-cadherin adherence junctions, and downregulation of its activity promotes the assembly of connexin43 gap junctions.20

The relatively quiescent keratocytes of the corneal stroma are activated during wound healing to proliferative fibroblasts and then to contractile myofibroblasts.21 TGF-ß1 can induce the phenotypic transition of fibroblasts to myofibroblasts. By removing TGF-ß1 and providing bFGF and heparin in the medium the myofibroblasts lose expression of {alpha}-smooth muscle actin ({alpha}-SMA) and regain the fibroblast phenotype.22 The transition of keratocytes to fibroblasts is associated with increased actin filament and stress fiber assembly, and the transition to myofibroblast is accompanied by synthesis of {alpha}-SMA and development of a more robust network of stress fibers.21 23 Rho/ROCK signaling regulates actin filament assembly and is probably a key element in these keratocyte transitions. Moreover, ROCK-I is essential for centrosome positioning,24 suggesting a direct and stringent relationship between ROCK and mitosis. Interleukin (IL)-1{alpha}–dependent expression changes in corneal fibroblasts have been surveyed successfully using DNA microarrays.25 To determine the gene expression effects of Rho/ROCK signaling, we inhibited ROCK activity in corneal fibroblasts and myofibroblasts and used oligonucleotide microarrays to measure the resultant changes in mRNA levels.


   Methods and Materials
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 Abstract
 Methods and Materials
Results and Discussion
 References
 
Cell Culture and RNA Isolation
Human donor eyes deemed unsuitable for transplantation were obtained from Center for Organ Recovery and Education (CORE; Pittsburgh, PA), in accordance with the provisions of the Declaration of Helsinki for research involving human tissue. Corneal fibroblasts cultured from stromal tissue explants from the donor corneas26 were grown in Dulbecco’s modified Eagle’s medium supplemented with Ham’s nutrient mixture F-12 (DMEM-F12; Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (Atlanta Biologicals Inc., Norcross, GA). Confluent cultures in passages 2 to 4 were subcultured in three sets of 60-mm dishes at a density of 5000 cells/cm2. After 24 hours in culture, cells were grown in DMEM-F12 with additions as detailed in Table 1 . In serum, growth-inducing factors predominate over growth-suppressing factors, and so, to induce the myofibroblast phenotype, the serum component was decreased to potentiate the effect of TGF-ß1. After 12 hours of Y-27632 (Tocris Cookson Inc., Ellisville, MO) or vehicle treatment, the cells in one dish from each experimental set were immunostained for {alpha}-SMA (100 cells were examined in three different fields) and the other dish(es) used for RNA extraction. No Y-27632–dependent apoptosis was apparent by microscopy. Total RNA was extracted from these cells (RNeasy; Qiagen, Valencia, CA). Yields were 11 to 14 µg RNA per confluent 60-mm culture dish, and 6 to 15 µg total RNA was used for sample preparation. Across the nine sample pairs, total RNA extracted from Y-27632–treated cells was 99% ± 14% (mean ± SD) of that extracted from matched controls, indicating no cell loss.


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  		TABLE 1. Stromal Cell Culture and Treatment

 
cRNA Preparation and Array Hybridization
cRNA preparation and array hybridization were performed according to the microarray technical literature (Affymetrix, Santa Clara, CA). Briefly, the mRNA component (≥0.5 µg) of extracted total RNA was reverse-transcribed (SuperScript II; Invitrogen-Life Technologies, Gaithersburg, MD) in the presence of a T7-(dT)24 primer (Genset Corp., La Jolla CA). The cDNA product was purified using phenol-chloroform extraction followed by ethanol precipitation of the cDNA from the aqueous phase. The cDNA was transcribed in vitro in the presence of biotin-labeled ribonucleotides (BioArray; Enzo, Farmingdale, NY). The biotinylated RNA product (≥35 µg) was separated from cDNA and other transcription components (RNeasy; Qiagen).

A portion (20 µg) of the biotinylated RNA was fragmented in a heat- and ion-dependent manner, and the fragments were hybridized overnight to one of the microarray (GeneChip; Affymetrix) formats. The chip was washed and developed by sequential treatment with streptavidin-phycoerythrin, anti-streptavidin antibody (conjugated to biotin), and then streptavidin-phycoerythrin. The GeneChip was scanned (ChipScanner; Agilent, Palo Alto, CA), and the collected data were processed and analyzed (MicroArray Suite [MAS] ver. 5.0; Affymetrix).

Microarray Samples
Human corneal stromal cells were derived from three separate donors. Each cell preparation was cultured under six different conditions (Table 1) without cross-donor mixing, yielding 18 unique samples. Two preparations of stromal cells (i.e., 12 samples) were analyzed with HU133A chips, and the remaining preparation (6 samples) was analyzed with HU95Av2 chips. Expression levels for each chip were normalized to the mean level (216) for all 18 chips. The HU95Av2 and HU133A formats contain 12,625 panels and 22,283 panels, respectively, with 9,843 panels showing good agreement between formats (Affymetrix technical literature). Panels measure both perfect match (signal) and mismatch (background) components. The MAS v5.0 software designates a transcript as present only if the perfect-match component is significantly (P < 0.05, single-tailed Wilcoxon signed-rank test) greater than the mismatch; otherwise, it is designated absent. Similarly, the software evaluates the significance of apparent changes between Y-27632–treated samples and matched controls, designating an increase or decrease only if P < 0.006. Our selection rules for three comparisons were as follows, with the less stringent requirements for six or nine comparisons in square brackets. For decreases, every control transcript must be present, and every Y-27632–treated sample must decrease relative to its control; every pair-wise ratio (i.e., Y-27632/uninhibited) must be ≤0.67, [≤0.8] and the mean of the pair-wise ratios (Y-27632/uninhibited) for three experiments must be ≤0.5 [≤0.67]. For increases, every Y-27632-treated transcript must be present and increase relative to its control; every pair-wise ratio must be ≥1.5 [≥1.25], and the mean of the pair-wise ratios (Y-27632/uninhibited) for three experiments must be ≥2 [≥1.5].


   Results and Discussion
Top
 Abstract
 Methods and Materials
 Results and Discussion
References
 
There was good correlation between Y-27632–treated cells and matched controls, with R2 = 0.977 ± 0.004 (mean ± SD, n = 3) for U95A chips and R2 = 0.976 ± 0.004 (mean ± SD, n = 6) for U133A chips. These data highlight the relatively small effect of Y-27632 and the excellent chip-to-chip reproducibility when similar samples are analyzed. They are comparable with the R2 (0.95) for chip-to-chip reproducibility previously reported,25 using corneal fibroblasts derived from a single tissue source. Comparisons between samples from different donor corneas which were treated identically in vitro gave regression coefficients of R2 = 0.939 ± 0.013 (mean ± SD, n = 6: U133A chips). Analysis of corneal epithelial cells on U95A chips (Harvey SAK, SundarRaj N, unpublished data; 2002) gave R2 = 0.970 ± 0.022 (mean ± SD, n = 3) for the Y-27632 effect and 0.900 ± 0.044 (mean ± SD, n = 6) for donor-to-donor variation. Taken together, these data show that donor-to-donor variation is greater than chip-to-chip variation for both chip formats and led us to select candidates on the basis of consistent multiples of change (x-fold) in pair-wise comparisons rather than requiring consistent absolute changes in expression.

Phenotypic Shift versus Y-27632–Driven Changes in Corneal Stromal Cells
In our dataset, the largest expression change was between activated fibroblasts (2%–5% cells immunostained for {alpha}-SMA) and myofibroblasts (>90% cells immunostained for {alpha}-SMA). This phenotypic shift was brought about by omission of exogenous bFGF plus heparin, a 10-fold decrease in serum-derived factors, and the addition of TGF-ß1 (Table 1) . The shift increased over 389 unique gene transcripts and decreased over 325, altering a relatively large number of transcripts (714/22,283 = 3.2% of those surveyed) over a wide range (205- to 0.007-fold). Preliminary analysis confirmed that this phenotypic shift includes several previously published TGF-ß–induced changes (Harvey SA, et al. IOVS 2003;44:ARVO E-Abstract 834). For example, microarray analysis of the effects of TGF-ß in human fetal lung fibroblasts yields 146 altered transcripts of the more than 6000 surveyed.27 Of these 146, 35 had exact matches in our data where chance would have predicted (146 x 714/22,283) or 4.7 matches. There were a further 11 inexact matches (i.e., different isoforms or family members).

In contrast to the 714 changes associated with phenotypic shift, only 66 unique transcripts (66/22,283 = 0.3%) were altered by Y-27632 treatment, and these changes were of smaller magnitude (6.6- to 0.31-fold). Transcripts decreased by Y-27632 were designated ROCK supported, and those increased by Y-27632 were designated ROCK suppressed. In Tables 2 3 4 and 5 , transcripts that are also altered by the shift from fibroblast to myofibroblast are identified by arrows that show whether the transcript is increased ({uparrow}) or decreased ({downarrow}) in myofibroblasts.


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  		TABLE 2. Global Y-27632 Effects in Stromal Cells

 

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  		TABLE 3. Y-27632 Effects in Baseline Fibroblasts: Stromal Cells Grown in 10% Serum

 

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  		TABLE 4. Y-27632 Effects in Activated Fibroblasts: Stromal Cells Grown in 10% Serum+bFGF+Heparin

 

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  		TABLE 5. Y-27632 Effects in Myofibroblasts: Stromal Cells Grown in 1% Serum and TGF-ß1

 
Y-27632–Driven Changes in Corneal Stromal Cells
Changes Common to All Stromal Groups.
We identified genes commonly controlled by ROCK signaling in all threestromal groups (Table 2) . For panels represented on both chip formats, combining all groups provided nine pair-wise comparisons and six pair-wise comparisons for panels present only on U133A chips. For these larger groups we used the less stringent ratio requirements, as described earlier. Moreover, we included candidates that had just one outlier (multiples of increase out of range, and/or transcript not present, and/or transcript level not changed). In Table 2 , ROCK-supported transcripts are broadly associated with cell cycle control and cytokinesis. We have hypothesized (see Fig. 8 in Ref. 28 ) that ROCK signaling upregulates the entry of cells into the S-phase of the cell cycle. ROCK supports both HMGB2, a nonhistone protein participating in the G1-to-S transition29 and ribonucleotide reductase subunit M2 (RRM2), which is the rate-limiting step in DNA synthesis in the S-phase and plays a crucial role in cell proliferation.30 Cyclin B both catalyzes entry into mitosis and promotes the S-phase,31 whereas cell division cycle 2/cyclin dependent kinase 1 (CDC2/CDK1) and cyclin kinase subunit, isoforms 1 and 2 (CKS) control entry into the M-phase through their interactions with cyclin B1.32

ROCK-supported control of mitosis occurs by PRC1, encoding a microtubule-associated protein that maintains the spindle midzone33 and is a phosphorylation substrate of cyclin B/CDC2,34 abnormal spindle-like, microcephaly associated/MCPH5/FLJ10517 (ASPM), which is a major determinant of the increased cerebral cortical size in humans,35 and rabkinesin6 (KIF20A/RAB6 interacting, kinesin-like protein/RB6K), which is essential for cytokinesis.36 37 RACGAP1 (MgcRacGAP for male germ cell RACGAP) identified38 as a GAP for Rac/Cdc42 shows progressive localization to the nucleus, mitotic spindle, and midbody in interphase, metaphase, and cytokinesis, respectively. RACGAP1is essential for cytokinesis,39 and when phosphorylated by aurora B kinase, it is functionally converted to a RhoGAP.40 Finally, the actin-associated protein encoded by ENC1 plays a regulatory role in the cytoskeletal reorganization and cell shape change that occur on differentiation from fibroblastic preadipocytes to spherical adipocytes.41

Although 10 µM Y-27632 causes 70% inhibition of thrombin-stimulated DNA synthesis in rat aortic smooth muscle cells,42 Y-27632 has no apparent effect on cell proliferation in FCS-stimulated human umbilical vein endothelial cells (HUVECs)43 or (until >200 µM) in renal fibroblasts.44 In Swiss 3T3 cells, inhibition of cytokinesis is detectable only at 30 µM Y-27632 or more. Twelve to 14 hours of treatment with 100 µM inhibitor delays G1-to-S progression considerably, but treated cells "catch up" by 32 hours.45 We saw a similar delay/catch-up pattern using 25 µM Y-27632, which together with the low inhibitor concentration and short exposure time probably explains why ROCK support of S-phase/mitosis transcripts appears weak.

Of six ROCK-suppressed transcripts, three are associated with cell signaling. Stromal cell derived factor (SDF-1/intercrine alpha/CXCL12), is an important chemotactic and angiogenic factor. Transcripts for both SDF-1 and its cognate receptor CXCR4 (NPYR3) have been detected in human corneal fibroblasts,46 and intact corneas have functional CXCR4 receptors as indicated by high-affinity SDF-1 binding. Cytosolic phospholipase A2 is a classic signal transduction enzyme that releases arachidonic acid from cell phospholipids and is activated by MAP kinase and by other ligand-receptor-invoked signaling pathways. Finally, type 2 phosphatidic acid phosphatases (PPAP2A) probably act as ectoenzymes to dephosphorylate signaling phospholipids such as lyso-phosphatidate, ceramide-1-phosphate, sphingosine-1-phosphate, and diacylglycerol pyrophosphate.47 PPAP2A expression is decreased in tumor cells48 suggesting that normally, it acts to decrease proliferation. If this is the case, ROCK suppression of the transcript will increase proliferation.

The cornea contains crystallin components49 (but see Ref. 50 ), and both aldo-keto reductase (AKR) and aldehyde dehydrogenase (ALDH) families are candidates for this role. In the present study, the AKR1 family members B1, C1, C2, C3, and B10 all were present at between 5 and 20 times the global mean expression level, whereas the "conventional" lens crystallins (ALDH3 members A1, B1, and B2) were absent. ROCK suppression of AKR1C2 suggests that Rho/ROCK signaling may have a role in regulating these presumptive AKR crystallins.

Active carbonic anhydrase II (CAII) binds to and doubles the activities of both the plasma membrane chloride/bicarbonate exchanger (AE1),51 and the Na+/H+ exchanger NHE1,52 suggesting that ROCK markedly suppresses the cells’ capacity to transport these ions. Finally, the hypothetical protein FLJ23191 fulfills stringent requirements for each phenotype (Tables 3 4 5) , making it the most important ROCK-associated transcript of unknown function.

Baseline Fibroblasts: Stromal Cells with no Added Growth Factors.
Table 3 shows that numerically, ROCK suppresses more transcripts than it supports. AKR1C2, PPAP2A, CAII, and FLJ23191 are all affected as previously discussed (Table 2) , whereas cell signaling is substantially modulated, with ROCK-suppressed transcripts encoding six extracellular mediators, a receptor diptych, and two protein kinase/phosphatase enzymes. Fibroblast growth factor 7/keratinocyte growth factor (FGF7/KGF) is a known paracrine mediator in the cornea,53 expressed in fibroblasts but not in corneal epithelial cells,54 and acting on corneal epithelial cells but not keratocytes through receptor FGFR2/KGFR.54 Consistent with this, KGFR is absent in stromal cells. Moreover, in corneal epithelial cells KGF is absent and KGFR is present (Harvey SAK, Anderson SC, SundarRaj N, unpublished data, 2003). TGF-ß2 is one of four transcripts for which the direction of Y-27632–driven change varies with culture conditions. In baseline fibroblasts expression is increased, whereas in myofibroblasts (Table 5) expression is decreased. Latent TGF-ß–binding protein 3 (LTBP3) is the second of these four transcripts. It shows moderate downregulation by Y-27632 (0.62-fold: not shown in Table 3 ) in baseline fibroblasts but is upregulated in myofibroblasts (Table 5) . LTBP3 is an extracellular matrix (ECM) protein that binds the small latent complex (SLC) of TGF-ß, and so reciprocal expression of LTBP3 and TGF-ß2 (the predominant endogenous TGF-ß isoform in the cornea) is consistent with homeostatic control of active TGF-ß2.

Both parathyroid hormone-like hormone (PTHLH/PTHrP: parathyroid hormone related peptide) and stanniocalcin 1 (STC1) control calcium and phosphate metabolism. PTHLH, like its prototype parathyroid hormone, stimulates bone resorption, renal tubular calcium reabsorption, 1,25-dihydroxyvitamin D3 (calcitriol) synthesis, and urinary excretion of phosphate. STC1 reversibly inhibits calcium L-channels55 and is upregulated in response to ischemia in the brain56 and in the failing heart.55 Taken together, these data suggest that STC can be upregulated in response to various types of stress and probably protects against intracellular Ca2+ overload. SCDGF-B/PDGF-D is the first known ligand specific for the ß isoform of the PDGF receptor.57 It potently transforms NIH/3T3 cells, eliciting stress fiber reorganization and increased proliferation rate.58 CXCL5 (SCYB5; small inducible cytokine subfamily B/ENA-78; epithelial-derived neutrophil-activating peptide 78) is a proinflammatory chemokine upregulated in cultured corneal stromal fibroblasts in response to IL-1{alpha} or TNF{alpha}.59

Transcripts are increased for endothelin (ET) receptor subtypes A and B (ETRA and ETRB). ET is the most potent vasoconstrictor currently known, and there is a rapidly growing body of literature implicating ROCK as a component in ET-dependent signaling. For example, Y-27632 inhibits ET-1-induced hypertrophy in neonatal rat cardiac myocytes.60

ROCK-suppressed signaling kinase/phosphatase enzymes are DYRK1A and PPP2R1B. The DYRK kinase family catalyze tyrosine-directed autophosphorylation but phosphorylate exogenous substrates at serine-threonine residues.61 Dynamin, known to be essential in cytokinesis62 is a substrate of DYRK1A.63 64 The protein phosphatase 2 (formerly protein phosphatase 2A) system comprises catalytic {alpha} and ß isoforms (PPP2CA and PPP2CB), either of which can associate with the regulatory {alpha} or ß isoform (PPP2R1A, PPP2R1B). The resultant core dimer can then interact with at least five distinct types of secondary regulatory subunits (for review, see Ref. 65 ). Of the four catalytic and primary regulatory subunits PPP2R1B is expressed at the lowest level in control cells, so that ROCK suppresses a limiting component of this complex signaling system.

ROCK-supported cell cycle control is represented by a transcript (HMGA1) encoding a high-mobility group nonhistone protein (HMG-I) that acts as an architectural transcription factor at mammalian promoters. HMG-I is phosphorylated by cdc2/Cdk1 in vivo,66 and this may contribute to the chromatin condensation that occurs at transition from G2 to mitosis (see HMGB2 in Table 2 ). ROCK-supported actin interactions are represented by smoothelin, which has a truncated actin-binding domain and associates with stress fibers. It was first described as a cytoskeletal protein specific for fully differentiated smooth muscle cells.67

Increased expression of intercellular adhesion molecule (ICAM-1/CD54/human rhinovirus receptor) in corneal disease68 increases leukocyte recruitment. As a consequence of their ICAM-1–mediated association, ROCK signaling occurs both in leukocytes69 and in endothelial cells.70 Moreover, ROCK-supported (Y-27632 inhibitable) ICAM-1 expression has been reported in cardiac allografts.71

Plasminogen activator inhibitor type (PAI)-1 inhibits the sequential activation of plasminogen and latent collagenase. ROCK support of PAI expression indicates that ROCK signaling inhibits remodeling of the ECM initiated by plasmin and collagenase. This is consistent with ROCK suppression of some ECM components in myofibroblasts (Table 5) .

Activated Fibroblasts: bFGF+Heparin-Treated Corneal Stromal Cells.
In bFGF-activated fibroblasts (Table 4) , the ROCK-supported transcripts ASPM (mitosis) and ENC1 (actin-associated) found previously (Table 2) are augmented by the mitosis-associated centromere protein F/mitosin (CENPF). As in baseline fibroblasts, ROCK suppresses CA2 and FLJ23191, and the cell-signaling components PTHLH and ETRA/ETRB (Table 3) . Additional signaling components suppressed by ROCK include two members of the TGF-ß superfamily, activin A and FLJ21195. Activin A and its cognate receptors are expressed in corneal fibroblasts, and exogenous activin A increases {alpha}2-SMA expression.72 ROCK suppression of activin A probably decreases these autocrine effects and favors the fibroblast over the myofibroblast phenotype. Moreover, hypothetical protein FLJ21195 is similar (54% identity plus 14% positives) to gremlin (CKTSF1B1), an endogenous antagonist of bone morphogenetic protein (BMP)-4 (a TGF-ß superfamily member) known to be important in renal fibrosis.73 The last cell signaling target is neuronal pentraxin I (NPTX1), a secreted glycoprotein that plays a role in remodeling of the nervous system.74 In Xenopus, the NPTX1 homologue is upregulated in regenerating epidermis.75 The transcriptional activator MyoD, which is essential to specification of muscle cell lineage, upregulates both NPTX1 and Id3 (inhibitor of DNA binding, a transcription corepressor) in undifferentiated myoblasts.76 Notably, in the present study Id4 was upregulated, suggesting a consistent relationship between NPTX1 and the Id family. These two components may contribute to ROCK suppression of myofibroblast conversion.

Two additional ROCK-suppressed transcriptional modulators are Nurr1 and NF-HEV. The transcriptional activator Nurr1 (nuclear receptor subfamily 4, group A, member 2: NR4A2/NOT/RNR1/HZF-3/TINUR) is a member of a superfamily that generally bind steroids or retinoids, but Nurr1 has no ligand binding site77 and its activity is probably modulated by other signal-transduction pathways such as MAP kinase.78 In bone cells, PTH induces Nurr179 suggesting that Nurr1 upregulation may be an autocrine response to elevated PTHLH. NF-HEV is a nuclear factor highly expressed in the specialized endothelium of high endothelial venules.80 The canine homologue DVS27 is expressed in (IL-1–stimulated) cultured smooth muscle cells,81 suggesting that ROCK suppression of this factor plays a role in suppressing myofibroblast conversion.

Tumor necrosis factor-{alpha}–induced protein 6 (TSG-6, TNFAIP6), is a member of the family of hyaluronate binding proteins, closely related to the adhesion receptor CD44 and often associated with extracellular matrix remodeling (for review, see Ref. 82 ).

Myofibroblasts: TGF-ß1/1% Serum-Treated Corneal Stromal Cells.
Upregulation of {alpha}2-SMA is diagnostic of the shift to myofibroblast phenotype, and in the present study this shift caused an 8.3-fold increase in {alpha}-SMA transcript levels, which was attenuated by Y-27632. The induction of {alpha}-SMA during renal fibrosis in vivo44 is decreased by Y-27632, which abrogates the TGF-ß1–induced formation of stress fibers in PC-3U human prostate carcinoma cells in vitro.83 In our hands, another SMA isoform ({gamma}2) was significantly downregulated (0.29-fold) by TGF-ß1 treatment, but remained susceptible to further downregulation by Y-27632.

Because two components of the ROCK-supported triad cyclin B1+CDC2+CKS2 (see Table 2 ) appear in Table 5 , we reviewed cyclin B1 and found that Y-27632 reduced its expression to 0.51 ± 0.13 of untreated levels. Thus, ROCK again supports the coordinated expression of all three components. ROCK facilitation of the M-phase includes support of DNA topoisomerase II{alpha}, which catalyzes the adenosine triphosphate (ATP)–dependent passage of one double-stranded DNA molecule through a transient break in another and is critically important for chromosome condensation and segregation. ROCK modulation of cytokinesis may also occur by support of GBP1, a member of the dynamin62 superfamily, which is largely responsible for the anti-proliferative response to inflammatory cytokines.84 Dual specificity phosphatase 6/MAP kinase phosphatase 3 (DUSP6/MPK3) contributes to G1 cell cycle arrest and apoptosis,85 therefore DUSP6 suppression by ROCK facilitates normal proliferation.

In myofibroblasts, ROCK supports TGF-ß2 expression and suppresses LTBP3 expression, changes opposite to those seen in baseline fibroblasts (Table 3) . Because TGF-ß2 is the principal endogenous isoform of TGF-ß, ROCK signaling supports autocrine maintenance of the myofibroblast phenotype. Moreover, ROCK suppresses the expression fibroblast growth factor receptor 1 (FGFR1, the receptor for bFGF/FGF2) decreasing sensitivity to bFGF and again maintaining the myofibroblast phenotype. Corneal epithelial and endothelial cells show a mitogenic response to hepatocyte growth factor (HGF), whereas stromal cells do not; however, transcripts for both HGF and its receptor (MET) occur in all three cell types.53 In our hands, ROCK supported MET and thereby a presumptive nonmitogenic stromal response.

As in baseline fibroblasts (Table 3) , ROCK suppresses both a member of the phosphatidic acid phosphatase type 2A superfamily, lipid phosphate phosphatase-related protein type 2a/FLJ13055 (LPPR2), and a candidate crystallin (AKR1B10). Myofibroblasts actively elaborate a wide range of ECM components, and ROCK suppresses a subset of these: biglycan, perlecan (heparan sulfate proteoglycan 2), agrin, adlican, collagen VII {alpha}1, and E2IG4.86 Finally, NAD(P)H-quinone oxidoreductase 1/cytochrome b5 reductase (NQO1) is widely distributed in the human eye, with strong immunostaining in keratocytes.87 NQO1 is bifunctional in response to oxidative stress. It has a cytoprotective, antioxidant role in vivo88 89 but also stabilizes the tumor suppressor p53,90 suggesting that it may trigger apoptosis in extreme conditions. ROCK suppression of NQO1 in myofibroblasts may decrease their vulnerability to apoptosis, relative to fibroblasts.

In summary, cytokines and growth factors are involved in corneal wound healing. Because bFGF and TGFß are expressed by both corneal epithelial cells and fibroblasts, their effects on corneal stromal cells during wound healing are both autocrine and paracrine (for review see Refs. 91 , 92 ). Quiescent stromal cells are activated during wound healing to become fibroblasts and myofibroblasts, the latter expressing {alpha}-SMA. Both these phenotypes have robust networks of actin filaments. Although one of the main functions of Rho/ROCK signaling is the assembly of stress fibers, other cellular processes are also regulated.93 The present study clearly indicates that Rho/ROCK signaling has both global effects on corneal stromal cell gene expression (e.g., cell cycle control and mitosis) and phenotype-specific effects. Given the number of panels that are separately altered by Y-27632 and the phenotypic shift from fibroblast to myofibroblast (66 and 714, respectively), random interaction between these conditions should yield only approximately two panels (66 x 714/22,283), which respond to both. Twenty-seven transcripts (see Tables 2 3 4 5 ) showed interaction between these two conditions, ~13-fold higher than predicted. This unexpectedly high number underlines the phenotype-specific importance of ROCK. For example, because TGF-ß2 is the predominant endogenous TGF-ß isoform in the cornea,94 ROCK suppression of TGF-ß2 in baseline fibroblasts attenuates autacoid TGF-ß–mediated conversion to myofibroblasts. However, if paracrine-mediated conversion occurs, ROCK accelerates it by supporting myofibroblast output of autacoid TGF-ß2. During wound healing, endogenous cytokines and growth factors modulate Rho/ROCK signaling. The present study shows that ROCK reciprocates by orchestrating both the cellular synthesis of mediators (TGF-ß2, PDGF-D, KGF, PTHLH, SDF-1 and stanniocalcin) and the cellular responses (through changes in their cognate receptors) to FGF, HGF, and ET.


   Acknowledgements
 
The authors thank Jean-Paul Vergnes and Paul R. Kinchington for assistance in initiating the study.


   Footnotes
 
Supported by National Eye Institute Grants EY03263 (NS), EY05945-15S1 (Robert L. Hendricks), and CORE Grant EY08098 to the Department of Ophthalmology; the Eye and Ear Foundation of Pittsburgh, and Research to Prevent Blindness.

Submitted for publication November 7, 2003; revised January 28 and March 11, 2004; accepted March 23, 2004.

Disclosure: S.A.K. Harvey, None; S.C. Anderson, None; N. SundarRaj, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Stephen A. K. Harvey, Department of Ophthalmology, University of Pittsburgh, EEINS 911, 203 Lothrop Street, Pittsburgh, PA 15213-2588; harveysa{at}msx.upmc.edu.


   References
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 Abstract
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 References
 

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