A Rho-associated protein kinase: differentially distributed in limbal ...

1266 Reports IOVS, June 1998, Vol. 39, No. 7

A Rho-Associated Protein Kinase:Differentially Distributed inLimbal and Corneal EpitheliaNirmala SundarRaj, Paul R. Kinchington,Howard Wessel, Brad Goldblatt, John Hassell,Jean-Paul Vergnes, and Susan C. Anderson

PURPOSE. The authors have developed monoclonal antibod-ies (mAbs) to characterize the sequential biochemicalchanges in corneal epithelial cells after they differentiatefrom stem cells, located in the limbus, and migrate centrip-etally to follow the pathway of terminal differentiation. Thepurpose of this study was to identify a protein (recognizedby mAb HE1/1 IF) with increased expression associated withthe transition of the limbal epithelium to corneal epithelium.

METHODS. The distribution and identification of the pro-tein(s) were performed using an indirect immunohistochem-ical staining technique and a western blot analysis, respec-tively. A rabbit corneal epithelial cDNA library, constructedin the Uni-Zap XR vector, was screened with mAb HE1/11Fto select cDNA clones expressing polypeptide(s) recognizedby this mAb. Additional overlapping cDNA clones were ob-tained from a primer extension cDNA library to determinethe sequence of die complete open reading frame encodingthe protein recognized by mAb HE1/11F.

RESULTS. Rabbit corneal epithelium exhibited strong im-munostaining with mAb HE1/11F, however, the limbalepithelial cells stained weakly. HE1/11F recognized 160-kDa (HEBM1) and 100-kDa (HEBM2) polypeptides in thecorneal epithelial extracts. The amino acid sequence ofthe protein deduced from the nucleotide sequence of thecDNA exhibited a close homology to that of a RhoA(Ras-related small GTPase)-associated serine-threonine ki-nase (ROCK-I or Rho-associated coiled-coil kinase). A 160-kDa RhoA-binding polypeptide with a molecular masssimilar to that of HEBM1 and ROCK-I was detected in thecorneal epithelial extracts. These findings strongly sug-gested that HEBM1 was rabbit ROCK-I. The identity ofHEBM1 was further confirmed from the reactivity of mAbHE1/11F with ROCK-I immunoprecipitated from rabbitcorneal epithelial extracts using anti-ROCK-I antibodies.

CONCLUSIONS. The increased expression of a protein identi-fied as ROCK-I from cDNA analyses is associated with rabbitcorneal epithelial differentiation and transition from the lim-bal to corneal surface. Therefore, a RhoA signaling pathwayis likely to be associated with corneal epithelial differentia-tion (maturation). A close homology among the cDNA se-

From the Department of Ophthalmology, University of PittsburghSchool of Medicine, Pennsylvania.

Supported in part by the Eye and Ear Foundation, a grantDCB910009P from Pittsburgh Supercomputing Center, and NationalInstitutes of Health grants EYO3263 and core grant EYO8098.

Submitted for publication November 12,1997; revised January 21,1998; accepted March 10, 1998.

Reprint requests: Nirmala SundarRaj, Department of Ophthalmol-ogy, Eye and Ear Institute, 203 Lothrop Street, Pittsburgh, PA 15213-

quences of rabbit, mouse, rat, and human ROCK-I indicatesthat this RhoA-associated kinase is a well-conserved protein.(Invest Ophthalmol Vis Set. 1998;39:1266 -1272)

Corneal epithelium is a self-renewing tissue and is main-tained by the migration of peripheral cells centripetally

and the division of basal cells followed by their migrationapically. A variety of observations indicate that the cornealepithelial stem cells are located in the limbus (see Refs. 1 and2 for reviews). To understand the mechanisms of cornealepithelial differentiation (maturation), it is important to gain aninsight into the biochemical changes associated with differentstages of corneal epithelial differentiation. Schermer et al.3

observed that a 64-kDa keratin (K3) expressed by differentiatedcorneal epithelial cells is not expressed in a subpopulation ofcells, possibly the stem cells, in the limbus. Several otherbiochemical differences in the limbal and corneal epithelialcells have since been reported (see Refs. 1 and 2 for reviews).

In the present study, monoclonal antibodies (mAbs) weredeveloped for use as probes to identify proteins that are dif-ferentially expressed in the corneal and limbal epithelial cells.In rabbit, one of these mAbs, HE1/11F, recognized protein(s)whose increased expression, as judged from immunostaining,was found to be associated with the migration of limbal epi-thelial cells over the corneal surface. Biochemical and cDNAanalyses identified a protein, recognized by mAb HE1/11F, asthe rabbit equivalent of the RhoA (Ras-related small GTPase)-binding serine-threonine kinase containing a large coiled-coildomain (ROCK-I or Rho-associated coiled-coil kinase), whichwas recently discovered in rat, mouse, and human nonoculartissues.4"6 This finding suggests that RhoA-GTPase signalingpathways involving ROCK-I are important in limbal to cornealepithelial phenotypic transition.

METHODS

Tissue and Tissue Culture

New Zealand white rabbits (6-8 weeks old) were killed usingan intravenous injection (Beuthanasia; Shering, Kenilworth,NJ). The corneas, with 1- to 2-mm adjacent sclera, were ex-cised, cut into half, and either embedded and frozen in com-pound (Tissue-Tek II OCT; Miles Laboratory, Elkhart, IN) at— 70°C for immunohistochemical analyses or used immediatelyfor tissue culture according to the method of Ebato et al.7

Primary explant cultures were passaged once or twice andused for extracting RNA for the construction of a cDNA library.All procedures involving rabbits were performed in compli-ance with the ARVO Statement for the Use of Animals inOphthalmic and Vision Research.

Antibodies and Immunostaining

The mAb, designated HE 1/1 IF, was selected from a panel ofmouse mAbs developed against limbal and corneal epithelialantigens using a plasma membrane-enriched fraction of hu-man corneal epithelial cells harvested from donor human cor-neas as the immunogens.8 Hybridomas, secreting mAbs againstcorneal epithelial antigens, were selected by immunohisto-chemical staining of cryostat sections of rabbit corneas.8 Oneof the hybridomas secreted the HE1/11F mAb and is described

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IOVS, June 1998, Vol. 39, No. 7 Reports 1267

in the present study. HE1/1 IF was characterized as an IgM typewitli K light chains. The hybridoma was either grown in tissueculture or in ascites form in balb/C mice. The antibody wasaffinity purified using anti-mouse IgM affinity gel (Cappel; Or-ganon Teknika, Durham, NC).

For immunohistochemical analyses,8 cryostat sections offrozen rabbit corneas were transferred onto gelatin-coated mi-croscope slides. The sections were reacted with appropriateconcentrations (0.5-1 jug/ml) of mAb HE1/11F or anothermouse IgM antibody (negative control) against a human-spe-cific antigen. The binding of the primary antibodies was de-tected by indirect immunofluorescence using fluorescein iso-thiocyanate or rhodamine-conjugated, goat anti-mouse IgMantibodies (Cappel, Organon-Teknika) at 1/100 dilution. Tostudy the distribution of ROCK-I, affinity-purified polyclonalgoat antibodies K-18 (5 /Ltg/ml) against a peptide correspond-ing to amino acids 1318 through 1337 at the carboxyl terminusof human ROCK-I (Santa Cruz Biotechnologies, Santa Cruz, CA)and fluorescein isothiocyanate-conjugated, rabbit anti-goatIgG antibodies (Cappel, Organon Teknika) at a 1:100 dilutionwere used as the primary and secondary antibodies, respec-tively.

Immunochemical and Biochemical Analyses of anAntigen Recognized by Monoclonal AntibodyHE1/11F

Comeal epithelium, scraped from 50 frozen rabbit eyes (PelFreez, Rogers, AR), was suspended in 5 ml buffer A (0.02 MTris-HCl [pH 8.0], 0.15 M NaCl, 0.1% sodium deoxycholate,0.1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS]) andprotease inhibitors (1.0 raM phenylmethylsulfonyl fluoride, 10mM EDTA, 2 raM n-ethyl maleimide, and 1 mg/ml pepstatin),homogenized with a manual tissue grinder (Wheaton, Millville,NT) using approximately 30 strokes, and then centrifuged atlOO.OOQg for 30 minutes at 4°C. For immunoblotting,9 proteinsin the supernatants were denatured in SDS sample buffer,separated on 7% SDS-polyacrylamide gel electrophoresis(PAGE) and electrophoretically transferred to a polyvinylidinedifluoride membrane (Millipore, Bedford, MA). Blots were im-munoreacted with mAb HE1/11F (0.5 fxg/mi). Horseradishperoxidase-conjugated, rabbit anti-mouse IgM (Calbiochem-Novabiochem, La Jolla, CA) antibodies (1:500 dilution) wereused as the secondary antibody to detect the binding of mAbHE1/11F. Blocking buffer (BLOTTO) containing 0.1%Tween-20 was used, and the reagent used for detection ofhorseradish peroxidase was either 4-chloro-l-naphthol (Bio-Rad, Hercules, CA) or chemiluminescence reagent (ECL; Am-ersham Life Science, Arlington Heights, IL).

Immunoprecipitation of ROCK-I

The protocol recommended by Santa Cruz Biotechnologies wasused to immunoprecipitate ROCK-I from rabbit comeal epithelialextracts in buffer A. Twenty microliters K-18 (200 peg/ml) and 20ixl protein G-agarose beads (Sigma Chemical, St. Louis, MO) wereused to immunoprecipitate ROCK-I from 2 ml epithelial extract.ROCK-I bound to the beads was extracted in SDS-PAGE samplebuffer by boiling the sample for 3 minutes, the beads wereremoved by centrifugation and the supernatants containingROCK-I were used for SDS-PAGE followed by immunoblot orRlioA binding analyses. Glutathione £transferase-RhoA-[35S]GTP7Sbinding to the blots was analyzed according to Matsui et al.10

MW A B C D

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III ~ ^FIGURE 1. OD Western blot analyses of monoclonal antibody(mAb) HEl/llF-reactive proteins. Soluble proteins extractedfrom rabbit corneal epithelial cells in culture (lanes A, Q ortissues (lanes B, D) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onpolyvinylidine difluoride membrane and either stained with Coo-massie blue (lanes A, B) or probed with mAb HE1/11F (lanes QD) followed by horseradish peroxidase-conjugated, anti-mouseIgM and chemiluminescence reagent (ECL) for detection. Note inlanes C and D: polypeptides with approximate molecular massesof 160 kDa and 100 kDa reacted with mAb HE1/11F. (IT) RhoA-GTP binding protein. Blots of the soluble proteins (similar tolanes C and D in I) were probed with mAb HE1/1 IF (lanes A, B)and identical blots were probed with [35S]GTPyS- bound gluta-thione S-transferase-RhoA (lanes C, D). Note a 160-kDa polypep-tide that binds radiolabeled RhoA-GTPyS (lanes C, £>), migratingidentically to mAb HEl/llF-reactive polypeptide (lanes A, B).(Ed) Western blot analyses of Rho-associated coiled-coil kinase(ROCK-I) immunoreactivity with mAb HE1/11F. Western blots ofthe soluble protein from rabbit corneal epithelial extracts, probedwith goat anti-ROCK-I antibodies (K-18) (lane A) and mAb HE1/11F (lane Q. Western blots of immunoprecipitated ROCK-I, fromsoluble protein extracts of rabbit corneal epithelium, wereprobed with K-18 (lane B) and mAb HE1/1 IF (lane D). Note, the160-kDa polypeptide (ROCK-I), immunoprecipitated with'anti-ROCK-I antibody, reacted with mAb HE 1/1 IF (lane D).



Construction and Screening of a Rabbit CornealEpithelial cDNA LibraryTotal RNA was extracted from approximately 1 X 108 culturedcorneal epithelial cells (25-mm X 100-mm dishes) in second passage,using the guanidine isothiocyanate-phenol-chloroform extractionprocedure.'' Poly(A) mRNA was isolated by affinity chromatographyon oligo(dT>cellulose (Pharmacia Biotech, Uppsala, Sweden) andused as a template for the construction of an oligo(dT>primed Uni-ZAP XR cDNA library, using a packaging extract (Gigapak II; Strat-agene, La Jolla, CA) according to the manufacturer's protocol. Ap-proximately 200,000 plaque-forming recombinants from this librarywere transduced into Escherichia coli PLK-F cells and plated on agardishes. Fusion protein expression was induced by placing isopropyl/3-i>thiogalactopyranoside-impregnated membranes (Duralose; Strat-agene) in each dish. Tlie membranes were immunoscreened withmAb HE1/11F vising a standard procedure. Plaques containing mAbHE1/1 IF- binding phage were selected, and the phage were purifiedand characterized by insertion size. The largest cDNA insert, 1.48 kbin length, was sequenced. A synthetic 28-base polynucleotide (nu-cleotides 1915-1942) at the 5' end of tlie initial clone was used torescreen and select additional clones in tlie library by hybridization.From the largest of the new clones, an insert 3.7 kb in length thatincluded the sequence of the first clone was obtained. A primerextension library was developed using a 25-bp primer containingsequences close to the 5' end of this clone (nucleotides 1344-1368)and tlie oligo(dT) primer. The library was screened with a primer(nucleotides 724-751) 592 bp upstream from the primer used forconstructing the library. From this screening, a cDNA clone (5.5 kbin length) was isolated and sequenced to identify the start codon andto unravel the sequence of die complete open reading frame (ORF).

DNA Sequencing and Sequence AnalysesThe pBluescript plasmid derivatives of the cDNA clones wererescued from Uni-ZAP XR by coinfecting the vector with f 1 -helperphage R408 using E. coli XL-1 Blue cells as the host. The rescuedphagemid from diese clones was purified and used as tlie se-quence template. The sequences of bodi strands were obtainedusing synthetic primers and the dideoxy nucleotide chain termi-nation procedure using a kit (Sequenase 2.0; US Biochemicals,Cleveland, OH). Tlie nucleotide sequences and deduced aminoacid sequences were analyzed for homology with published se-quences using die Pittsburgh Super Computing Center to accesstlie DNA and Protein Databases using software (GCG; GeneticComputer Group, Madison, WT) for the analyses.

RESULTS

Western Blot Analyses of the Proteins Recognizedby Monoclonal Antibody HE 1/1 IFTo identify the proteins recognized by mAb HE1/11F, rabbitcorneal epithelial extracts in buffer A were analyzed using thewestern immunoblot technique. As seen in Figure II, twopolypeptides with approximate molecular masses of 160 kDaand 100 kDa reacted with mAb HE1/11F and were designatedHEBM1 and HEBM2, respectively.

cDNA Clones and Sequence AnalysesFrom a rabbit corneal epithelial cDNA library constructed inthe Uni-Zap XR expression vector, five cDNA clones, encodingmAb HEl/llF-reactive polypeptides, were immunoselected.

The largest of these overlapping clones, clone 1, was se-quenced. The sequence did not contain either the start or thestop codon of the ORF. A larger clone (clone 6), 3 7 kb inlength, which hybridized with clone 1, was selected. Clone 6completely overlapped with clone 1 and extended further, atthe 5' and 3' ends. However, neither the start nor stop codonof the ORF was present in clone 6. Therefore, a primer exten-sion library was developed. A clone (clone X2) selected fromthis library was found to completely overlap with the first twoclones and contained the complete ORF (303-4365) encoding1354 amino acids. The sequence of this clone has been sub-mitted to GenBank (accession number U42424).

When compared with the published sequences in thedatabases, the nucleotide sequence and the deduced aminoacid sequence of the complete ORF of clone X2 showed over95% homologies with those of rat, mouse, and human Rlio-associated serine-threonine kinase (ROCK-I), a recently discov-ered RlioA-binding protein (Fig. 2). The protein encoded byclone X2 was, therefore, identified as the rabbit equivalent ofROCK-I. The rabbit ROCK-I sequence, like that from otherspecies, has several interesting domain structures and motifs(Fig. 2). The signature sequences of protein kinase adenosinetriphosphate (ATP)-binding region (amino acids 82-105) andthe serine-tlireonine protein kinase active site (amino acids194-206) are located at the amino terminus of the molecule,followed by a large coiled-coil domain (amino acids 423-1100)as determined by an algorithm developed by Lupas et al.12 Aleucine zipper motif (amino acids 941-962) is present withinthe coiled-coil region. A pleckstrin homology domain (aminoacids 1118-1317) containing a phorbol ester-diacylglycerolbinding domain (amino acids 1228-1283) is present at thecarboxyl terminus.

Analyses of RhoA-Binding ProteinsThe deduced molecular mass (158 kDa) of rabbit ROCK-Iencoded by clone X2 was similar to that of HEBM1, as judgedfrom its migration in SDS-PAGE. This finding suggested thatHEBM1 is most likely rabbit ROCK-I. Therefore, the ligandbinding assay was performed to determine whether a RlioAbinding protein of the same molecular mass was present in thecorneal epithelial extracts. The Al60-kDa protein, which mi-grated identically to HEBM1 in SDS-PAGE, was found to bindglutathione S-transferase-RhoA-[35S]GTPyS and not RlioA-[35S]GDP/3S (Fig. 1, Part II). This finding strongly suggestedthat HEBM1, the RlioA binding protein and the protein en-coded by clone X2, were the same protein.

Reactivity of Monoclonal Antibody HE1/11F withROCK-IThe proteins, immunoprecipitated from the rabbit corneal ep-ithelial extract using K-18 antibodies, were analyzed for theirreactivity with HE1/11F and K-18 antibodies using the westernblot technique. Both the antibodies reacted with a polypeptide(ROCK-I) with an approximate molecular mass of 160 kDa (Fig.1 III). This finding confirmed that mAb HE1/11F recognizesROCK-I in the rabbit corneal epithelial extract.

Distribution and Characterization of Protein(s)Recognized by Monoclonal Antibody HE1/11F,ROCK-I, and K3 KeratinBecause K3 keratin has been used as a marker for differentiatedcorneal epithelial cells and the absence of keratin as the marker



HEBMHumRMusRRatR

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HEBMHumRMusRRatR

HEBMHumRMusRRatR

HEBMHumRMusRRatR

HEBMHumRMUSR

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HEBMHumRMusRRatR

HEBMHumRMusRRatR

HEBMHumRMusRRatR

HEBMHumRMusRRatR

HEBMHumRMusRRatR

HEBMHumRMusRRatR

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RatR

MSTGDSFETR FEKgJDNLLRD PKSEVNSDCLMSTGDSFETR FEK$|DNLLRD PKSEVNSDCLMSTGDSFETR FEK§DNLLRD PKSEVNSDCLMSTGDSFETR FEKIDNLLRD PKSEVNSDCL81 ATP bindingVIGRGAFGEV QLVRHKSTRK VYAMKLLSKFVIGRGAFGEV QLVRHKSTRKVIGRGAFGEV QLVRHKSTRKVIGRGAFGEV QLVRHKSTRK161LVNLMSNYDV PEKWARFYTALVNLMSNYDV PEKWARFYTALVNLMSNYDV PEKWARFYTALVNLMSNYDV PEKWARFYTA241ISPEVLKSQG GDGYYGRECDISPEVLKSQG GDGYYGRECDISPEVLKSQG GDGYYGRECDISPEVLKSQG GDGYYGRECD321EVRLGRNGVE EIKRHLFFKNEVRLGRNGVE EIKRHLFFKNEVRLGRNGVE EIKRHLFFKNEVRLGRNGVE EIKRHLFFKN401SNRRYLS|AN PN|NR|SSN|SNRRYLSiAN P ^ N R | s S N |SNRRYLP|AN AS|NR|SSN|SNRRYLP|AN PS|NR|SSN|481VSQIEKEKML LQHRINEYQRVSQIEKEKML LQHRINEYQRVSQIEKEKML LQHRINEYQRVSQIEKEKML LQHRINEYQR561AVRLRKSHTE MSKSj|SQLESAVRLRKSHTE MSKs||SQLESAVRLRKSHTE MSKs|iSQLESAVRLRKSHTE MSKsfsQLES641LK|NLE||EG ERKEAQDMLNLK|NLE|||EG ERKEAQDMLN

LK|NLE|§|EG ERKEAQDMLN

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KEEREAREKA ENRW||EKQ801KGLEKQMKQE INTLLEAKRLKGLEKQMKQE INTLLEAKRLKGLEKQMKQE INTLLEAKRLKGLEKQMKQE INTLLEAKRL881LQNEKETL|T QL D L A E T K A E

LQNEKETLJ§T QLDLAETKAELQSEKETL|T QLDLAETKAEL Q S E K E T L I * QLDLAETKAE

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LDGLDALVYDLDGLDALVYDLDGLDALVYDLDGLDALVYD

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LDFPALRKNK NIDNFLSRYKLDFPALRKNK NIDNFLSRYKLDFPALRKNK NIDNFLSRYKLDFPALRKNK NIDNFLSRYK

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CSMLDVDLKQCSMLDVDLKQCSMLDVDLKQCSMLDVDLKQ

LEFELAQLTKLEFELAQLTKLEFELAQLTKLEFELAQLTK

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FIGURE 2. (Top) Comparison of the deduced amino acid sequences of the rabbit protein (HEBMl) and Rho-associated coiled-coil kinase(ROCK-I) from human (HumR),4 mouse (MusR),5 and rat (RatR).6 The amino acid sequences were deduced from nucleotide sequencesof corresponding cDNAs. Underlined are the following motif structures present in HEBMl and also in human, mouse, and rat ROCK-I:ATP binding, signature sequence of ATP-binding region (amino acids 82-105) of protein kinases; Ser-thr kinase, signature sequence ofserine-threonine protein kinases active-site (amino acids 194-206); PKAP, cAMP-dependent protein kinase phosphorylation site (aminoacids 527-530); TYP, tyrosine phosphorylation site (amino acids 596-604, 906-913, and 967-973); LZ, leucine zipper pattern (aminoacids 941-962); PH, pleckstrin homology domain (amino acids 1118-1317); and CRD, cysteine-rich domain, a phorbol ester-diacylg-lycerol binding site (amino acids 1228-1283). The differences in the sequence of ROCK-I between the species are highlighted. (Bottom)Schematic representation of HEBMl (similar to ROCK-I in other species) shows the major domain structures in the molecule.



1041 1117HEBM LNQEREKFNQ MWKHQKELN DMQAQLVEEC §!HRNELQMQL ASKESDIEQL RAKL|DLSDS TSVASFPSAD ETDENLP. . .HumR LNQEREKFNQ MWKHQKELN DMQAQLVEEC |HRNELQMQL ASKESDIEQL RAKL|DLSDS TSVASFPSAD ETD|NLP. . .MusR LNQEREKFNQ MWKHQKELN DMQAQLVEEC |HRNELQMQL ASKESDIEQL RAKL|DLSDS TSVASFPSAD ETD|NLP...RatR LNQEREKFNQ MWKHQKELN DMQAQLVEEC |HRNELQMQL ASKESDIEQL RAK L | D L S D S TSVASFPSAD ETD|NLP]|||

1118 PH 1185HEBM JSRIEGWL SVPNRGNIKR YGWKKQYWV SSKKJLFYND EQDKEQSNPS MVLDIDKLFH VRPVTQGDVYHumR isRIEGWL SVPNRGNIKR YGWKKQYWV SSKKfiLFYND EQDKEQSNPS MVLDIDKLFH VRPVTQGDVYMusR $SRIEGWL SVPNRGNIKR YGWKKQYWV SSKK|;LFYND EQDKEQSSPS MVLDIDKLFH VRPVTQGDVYRatR f&XW&tt&t $$S.SRIEGWL SVPNRGNIKR YGWKKQYWV SSKKiLFYND EQDKEQSSPS MVLDIDKLFH VRPVTQGDVY

1186 PH CRD 1265HEBM RAETEEIPKI FQILYANEGE CRKDJIJEEitEPV QQlilEKTNFQN HKGHEFIPTL YHFPANCEAC AKPLWHVFKP PPALECRRCHHumR RAETEEIPKI FQILYANEGE CRKD|E|EPV QQfSEKTNFQN HKGHEFIPTL YHFPANC|AC AKPLWHVFKP PPALECRRCHMusR RAETEEIPKI FQILYANEGE CRKD§Ef|EPV QQiEKTNFQN HKGHEFIPTL YHFPANC|AC AKPLWHVFKP PPALECRRCHRatR RAETEEIPKI FQILYANEGE C R K D I E I E P V Q Q | E K T N F Q N HKGHEFIPTL YHFPANc|AC AKPLWHVFKP PPALECRRCH

1266 "" PH "• "• 1345

HEBM VK&JHRDHLDK KEDLIjgPCKV SYDVTSARDM LLLACJQDEQ KKWVTHLVKK IPKJJPSGFV RASPRTLSTR STANQSFRKVHumR VKj|HRDHLDK KEDLI|PCKV SYDVTSARDM LLLACigQDEQ KKWVTHLVKK IP K | | P S G F V RASPRTLSTR STANQSFRKV'MusR VK|HRDHLDK KEDLl|PCKV SYDVTSARDM LLLAC|QDEQ KKWVTHLVKK IPKf§|PSGFV RASPRTLSTR STANQSFRKVRatR V K | H R D H L D K KEDLI|PCKV SYDVTSARDM LLLAClQDEQ KKWVTHLVKK IPK§§PSGFV RASPRTLSTR STANQSFRKV

134 6HEBM VKNTSGKTS*HumR VKNTSGKTS.MusR VKNTSGKTS* .. . ..RatR VKNTSGKTS* ,,. _ . . . COlled-COlI

ser/thr kinase domain structure PH domain

LZ CRD

FIGURE 2. (Continued)

for stem cells in the limbus,3 the staining patterns with mAbHE1/11F and anti-K3 antibody (AE5) were compared usingserial tissue sections of the rabbit cornea with the surroundinglimbus. When reacted with anti-K3 antibody, bright fluores-cence staining was seen in all layers of corneal epithelium, andas expected, staining was absent in the basal cells of the limbus(Fig. 3A). In the consecutive serial tissue section, the cornealepithelium exhibited a strong fluorescence staining (Fig. 3B)with mAb HE 1/1 IF. The intensities of staining with mAb HE1/11F were abruptly decreased in the limbal epithelium startingat the limbal-corneal junction. In the next consecutive sec-tion, which was reacted with K-18 antibodies, the stainingpattern was similar to that seen with mAb HE 1/1 IF. A weakfluorescence was detectable in the basal cells in the limbus;however, it was more negligible then the bright fluorescencein the corneal epithelium (Pig. 3C).

DISCUSSION

We have developed a library of mAbs to characterize pheno-typic changes associated with the differentiation of cornealepithelium from the stem cells located in the limbus and theirfurther differentiation as they migrate centripetally and api-cally. One of these mAbs (HE1/11F) recognized two proteins,designated HEBM1 and HEBM2 (approximate molecularmasses of 160 kDa and 100 kDa, respectively), whose in-creased expression was associated with corneal epithelial dif-ferentiation. In the present study, cDNA clones were selectedfrom a rabbit corneal epithelial cDNA library using HEl/1 IF forimmunoscreening. The amino acid sequence deduced from thenucleotide sequence of cDNAs exhibited a close homologywith a recently discovered RhoA-associated serine-threoninekinase (ROCK-I). ROCK-I in rat, human, and mouse tissues andanother closely related kinase (ROCK-II or ROKa) were discov-

ered when three different groups of investigators were search-ing for RhoA-GTP-binding proteins.4"6 The molecular mass(158 kDa) of rabbit ROCK-I, determined from the deducedamino acid sequence, was comparable to that of HEBM1 (160kDa) estimated from its migration in SDS-PAGE. A RhoA-GTP-binding protein in the corneal epithelial extracts, migratedidentically to HEBM1. These findings strongly indicate that the160-kDa protein, HEBM1, was the same as ROCK-I, which wasidentified from cDNA analyses. The reactivity of HEl/1 IF withROCK-I immunoprecipitated using polyclonal ROCK-I-specificantibodies (K-18), further confirms the identity of HEBM1 to beROCK-I. The association of increased expression of ROCK-Iwith limbal to corneal epithelial transition was also confirmedimmunohistochemically using ROCK-I-specific antibodies.

Currently, the identity of HEBM2 is unknown. It is possi-ble that HEBM2 may be an alternatively spliced variant ofHEBM1 or an unrelated protein sharing a common epitopewith HEBM1. Western blot analyses suggest that HEBM2 isexpressed at significantly higher levels than HEBM1 in thecorneal epithelium. Therefore, HEBM2 possibly contributedmore to the bright fluorescence in the corneal epithelium, andthe absence (or low levels) of HEBM1 and HEBM2 were re-sponsible for the weak staining in the limbus. However, untilfurther analyses are conducted using specific probes, the iden-tity of HEBM2 and its association with corneal epithelial differ-entiation remain speculative.

The increased levels of ROCK-I in the corneal epithelialcells suggest that a RhoA GTPase signaling pathway involvingROCK-I is likely to be important in corneal epithelial differen-tiation and maintenance. The Rho (Ras homologous) family ofproteins belongs to the Ras superfamily of small GTP-GDPbinding proteins, and in the GTP-bound form, these proteinspossess GTPase activity (see Refs. 13 and 14 for reviews).RhoA, -B, and -C are closely related proteins, which have close



FIGURE 3. Indirect immunofluorescence staining. Serial cryostat sections of the rabbit corneaswith the surrounding limbus were immunoreacted with and K3-keratin mAb (AE5) (A); mAbHEl/llF (B); affinity-purified goat anti-Rho-associated coiled-coil kinase antibodies (K-18) (C); andgoat IgG (negative control) (D), followed by rhodamine-conjugated anti-mouse IgG (A), anti-mouseIgM (B), or anti-goat IgG (C. D) antibodies. A phase contrast micrograph of (C) is shown in (E).Arrows in (A) point to the junction between the limbal (K3-negative basal cells) and comeal(K3-positive basal cells) epithelia. In die serial sections, (B) and (C), note the transition fromnegligible to bright staining from the limbal to corneal epithelial cells. Scale bar, 50 /j,m.

homologies at the amino termini but differ significantly in theircarboxyl termini. RhoA, the most ubiquitous of the Rho family,is the best characterized. RhoA has been shown to regulateseveral cellular processes involving actin, including formationof focal adhesions and stress fibers, cell migration, cellularproliferation, changes in cell morphology, membrane ruffling,cytokinesis, cell aggregation, and smooth muscle contraction(see Refs. 13 and 14 for reviews).

Several enzymes of phospholipid metabolism includingphosphotidylinositoM-phosphate-5-kinase, phosphoinositide-3-ki-nase, and phospholipase D (see Refs. 13 and 14 for reviews) havebeen shown to be activated by RhoA in crude membrane prepa-rations of cell lysates of C3H fibroblasts. These results suggest thatRhoA regulates the actin cytoskeletal reorganization, possiblythrough the formation of phospholipid metabolites that may beinvolved in this process. RhoA has also been shown to regulateone or more genistein-sensitive tyrosine kinases. Although RhoAmay be a key regulator affecting many fundamental cellular pro-cesses, the direct interactions with the regulatory elements thatcause the effects have not been elucidated.

In the last 2 years, a search for the direct downstreamtargets for RhoA-GTP has identified several RhoA-binding pro-teins including ROCK-I. The kinase domain of ROCK-I is lo-cated at the amino terminus, which is followed by a largecoiled-coil domain. The Rho-binding domain is located at thecarboxyl terminus region of the coiled-coil domain. After thecoiled-coil region, there is a pleckstrin homology region,which may promote the interaction of ROCK-I with the cell

membrane. There is a cysteine-rich zinc finger-like motif withinthe pleckstrin homology domain at the carboxyl terminus ofthe molecule. Studies on the role of these kinases in theRho-signaling pathways are still in their infancy. A direct in-volvement of ROCK-I in stress fiber and focal adhesion assem-bly has not been reported. ROCK-I has several interestingmotifs, which could be important in its interaction with one ormore downstream targets in its signaling pathway. Becausemost studies on RhoA and ROCK-I functions have been con-ducted using either transformed or nontransformed cells lines,one will have to be careful in extrapolating the results to thoseof the normal tissues. ROCK-I signaling pathways are likely tohave important functions in corneal epithelial differentiation,maintenance, wound healing, and development, which will beexplored in our future studies.

Acknowledgments

The authors thank Robb Belak and Jane Wang for their technical assis-tance and Judy Smith and Geri Gutkowski for their secretarial assistance.

References

1. Tseng SC. Regulation and clinical implications of corneal epithelialstem cells. Mot Biol Rep. 1996;23:47-58.

2. Zieske JD. Perpetuation of stem cells in the eye. Eye. 1994;8(Pt2): 163-169.

3. Schermer A, Gatvin S, Sun T-T. Differentiation-related expressionof a major 64K corneal keratin in vivo and in culture suggests



limbal location of corneal epithelial stem cells . / Cell Biol. 1986;103:49-62.

4. Ishizaki T, Maekawa M, Fujisawa K, et al. The small GTP-binding proteinRho binds to and activates a 160 kDa Ser/Thr protein kinase homologousto myotonic dystrophy kinase./£MBQ 1996;15:1885-1893.

5. Nakagawa O, Fukisawa K, Ishizaki T, et al. ROCK-I and ROCK-II,two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBSLett. 1996;392:189-193.

6. Leung T, Chen XQ, Manser E, et al. The pl60 RhoA-binding kinase ROKalpha is a member of a kinase family and is involved in the reorganizationof the cytoskeleton. Mol Cell Biol 1996;l6:5313-5327.

7. Ebato B, Friend J, Thoft RA. Comparison of limbal and peripheralhuman corneal epithelium in tissue culture. Invest Ophthalmol VisSci. 1988;29:1533-1537.

8. Langer MG, SundarRaj CV, SundarRaj N. Corneal epithelial-specificcell surface antigen recognized by a monoclonal antibody. / Em-bryol Exp Morphol. 1986;94:l63-172.

9. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of pro-teins from polyacrylamide gels to nitrocellulose sheets: procedureand some applications. Proc Natl Acacl Sci USA. 1979;76:4350-4354.

10. Matsui T, Amano M, Yamamoto T, et al. Rho-associated kinase, anovel serine/threonine kinase, as a putative target for small GTPbinding protein Rho.JEMBO. 1996;15:2208-22l6.

11. Chomczynski P, Sachhi N. Single step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction. AnalBiochem. 1987;l62:156-159.

12. Lupas A, Van Dyke M, Stock J. Predicting coiled coils from proteinsequences. Science. 1991 ;252:1162-1164.

13- Ridley AJ. Rho-related proteins: actin cytoskeleton and cell cycle.Curr Opin Genet Dev. 1995;5:24-30.

14. Narumiya S. The small GTPase Rho-cellular functions and signaltransduction. / Biochem. 1996;120:215-228.

Expression of Nerve GrowthFactor Receptors on the OcularSurface in Healthy Subjects andduring Manifestation ofInflammatory DiseasesAlessandro Lambiase,1'5 Stefano Bonini,2

Alessandra Micera,1 Paolo Rama,5

Sergie Bonini,4 and Luigi Aloe1

PURPOSE. Recent studies have suggested the involvementof nerve growth factor (NGF) in the conjunctival inflam-matory process and in corneal epithelium proliferationand differentiation. To verify the hypothesis that NGFcould locally modulate the inflammatory and reparativeprocesses, the authors evaluated the expression of NGFhigh-affinity receptor on the ocular surface in normal andpathologic conditions.

METHODS. Ten conjunctival byopsies (obtained from threehealthy subjects, five patients affected by vernal keratocon-junctivitis [VKC], and two patients with cicatricial pemphi-goid [CP]) and five corneal specimens obtained from the EyeBank of Veneto (Italy) were evaluated. All specimens werehistologically stained, and immunohistochemistry was per-formed to identify the NGF high-affinity receptor (TrkA).

RESULTS. All tissues expressed immunoreactivity for NGFreceptors. In conjunctival specimens of healthy subjects,basal epithelial cells strongly expressed immunoreactivity

From the 'institute of Neurobiology, National Research Council,Rome; 2Division of Ophthalmology, Hospital of Venice "SS. Giovanni ePaolo"; 3Departement of Ophthalmology, University of Rome "TorVergata"; and ''Departement of Clinical Immunology and Allergology, IIUniversity of Naples, Italy.

Supported by a grant from P. F. Biotecnologie, CNR, SP5.Submitted for publication September 15, 1997; revised January 16,

1998; accepted January 27, 1998.Proprietary interest category: N.Reprint requests: Luigi Aloe, Research Director, Institute of Neu-

robiology, National Research Council, Viale Marx 15, 00132 Rome,Italy.

and, in the stroma, rare cells were immunopositive for TrkA.No significant difference in immunoreactivity was observed:n the conjunctival epithelium between healthy subjects andpatients with inflammatory conjunctival diseases, whereasthere were more immunopositive cells observed in the con-junctival stroma of VKC and CP patients than in the controls.The immunoreactivity in the cornea was confined to basalepithelial cells and endothelium.

CONCLUSIONS. The NGF receptor is present on the humanocular surface. The authors' data support the possibil-ity that NGF modulates ocular inflammation andcorneal epithelial proliferation and differentiationthrough its receptors. (Invest Ophthalmol Vis Sci. 1998;39:1272-1275)

To date, it has been demonstrated that the biologic actionof nerve growth factor (NGF), which is the best-charac-

terized member of the neurotrophin family, is not restrictedto cells of neuronal origin but also extends to cells of theimmune system.1 In particular, in vitro and in vivo studieshave shown that NGF induces mast cell differentiation, de-granulation, and mediator release; suppresses leukotrieneC4 formation from eosinophils; and stimulates T and Blymphocyte proliferation and activation.2 We have recentlydemonstrated a basal production of NGF by T-helper (Th)cell clones in vitro and an increased production of NGF byTh-2 after activation.3 Moreover, the presence of high levelsof circulating NGF in autoimmune and allergic human dis-eases has been demonstrated.4'5

Two recent studies have described the relationship be-tween NGF and the ocular surface: an in vitro study showingthat NGF induces proliferation and differentiation of rabbitcorneal epithelial cells6 and a clinical report showing an in-crease of NGF plasma levels in vernal keratoconjunctivitis(VKC) with a direct correlation between mast cell conjunctivalinfiltration and the NGF levels.5 To further verify the hypoth-esis of local involvement of NGF in the physiological andpathologic processes of the human ocular surface, we evalu-ated the presence of the NGF receptor on the cornea andconjunctiva of healthy subjects and in the conjunctiva duringmanifestation of inflammatory diseases.


A Rho-associated protein kinase: differentially distributed in limbal ...

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