|Molecular Vision 2004;
Received 21 July 2004 | Accepted 8 September 2004 | Published 14 September 2004
Differential expression of splice variants of chemokine CCL27 mRNA in lens, cornea, and retina of the normal mouse eye
Dolena R. Ledee,1 Jun
Chen,1 Leonardo H. Tonelli,2 Hiroshi Takase,1 Igal
Gery,1 Peggy S. Zelenka1
1National Eye Institute and 2National Institute of Mental Health, NIH, Bethesda, MD
Correspondence to: Peggy S. Zelenka, Ph.D., Building 7, Room 201, 7 Memorial Drive MSC 0704, Bethesda, MD, 20892-0704; Phone: (301) 496-7490; FAX: (301) 402-7682; email: email@example.com
Purpose: Constitutive expression of RNA sequences complementary to the chemokine CCL27 mRNA has been found in the normal mouse eye. This study examines the nature and location of these endogenous RNAs in ocular tissues.
Methods: Conventional RT-PCR, 5' RACE, and dideoxy DNA sequencing were used to examine the sequences of CCL27 related RNAs in the eye. Expression levels of specific RNAs were measured by real time PCR. Tissue distribution of RNA transcripts was determined by RT-PCR using RNA from microdissected tissues and by in situ hybridization with radiolabeled riboprobes.
Results: We detect 5 distinct splice variants derived from transcription of the CCL27 gene locus. The most abundant form codes for a non-secreted protein, PESKY, and is expressed in lens, cornea, and retina. Another variant corresponds to the mRNA of the secreted chemokine and is synthesized in the cornea, but not in retina or lens. The remaining splice variants are novel and may be eye specific, but have only short open reading frames (<50 amino acids). CCL27 transcripts are most abundantly expressed in the retina, as judged by in situ hybridization.
Conclusions: PESKY and other CCL27 splice variants of unknown function are widely expressed in ocular tissues. Analysis of CCL27 transcripts from lens, retina, and cornea indicates that mRNA for the secreted chemokine, CCL27, is endogenously expressed only in the cornea and may play a role in ocular immune responses involving CD4 lymphocytes in this tissue.
Ocular inflammation is responsible for approximately 10-15% of vision loss in the United States [1,2]. To intervene in this process it is necessary to understand the cellular and molecular mechanisms involved. To better understand these mechanisms, we have explored the role of chemotactic cytokines, or chemokines, which play a major role in the inflammatory process [3,4]. Chemokines are members of a large family of structurally related, heparin-binding, proteins that mediate cellular movement, particularly in inflammatory cells. The family is divided into four subfamilies on the basis of variations on a conserved cysteine motif. The two largest subfamilies each have four conserved cysteine residues, but differ in the presence or absence of a single amino acid insertion between the first two cysteines. Thus, in the CXC family the first two cysteines are separated by a single variable amino acid, while in the CC family these first two cysteines are juxtaposed. The two other subfamilies (C and CX3C) contain only single members (lymphotactin and fractalkine, respectively). Chemokine action is mediated by a family of seven-transmembrane, G protein coupled receptors, which are named according to the family of chemokines they bind (CCR, CXCR, etc.)
In a recent screen of mouse eye mRNAs we demonstrated constitutive expression of several chemokines, cytokines, and chemokine receptors not previously detected in the eye , including the chemokine CCL27, so named because it is a member of the CC family of chemokines and serves as a Ligand for the chemokine receptor, CCR10 . CCL27 (also called CTACK, ALP, and ESkine) is thought to be selectively expressed in the skin and to be responsible for migration of CD4 lymphocytes expressing the CCR10 chemokine receptor and the cutaneous lymphocyte-associated antigen (CLA) into this tissue . Although CCL27 has little activity on other immune and inflammatory cell types, an alternatively spliced variant, referred to as PESKY, is widely expressed and may have functions that are unrelated to the immune response [7,8]. PESKY and canonical CCL27 differ only in the first of three exons . While the CCL27 first exon encodes the classical chemokine signal peptide leading to secretion, the PESKY first exon lacks a signal peptide, but contains a nuclear localization signal, thus targeting the protein to the nucleus. In addition, the two splice forms have discrete tissue distributions, with CCL27 expressed predominately in skin and placenta and PESKY in the testes and brain .
In this study we have investigated the expression pattern of CCL27 and its alternatively spliced variants in ocular tissues, using conventional and real-time RT-PCR, and in situ hybridization. We have found that PESKY is the most abundant CCL27 splice product in the eye, although the fully spliced chemokine mRNA and two novel splice products are also present. Comparison of RNA from retina, cornea, and lens detected the fully spliced CCL27 chemokine mRNA only in the cornea, whereas PESKY was found in all three tissues.
All animal studies were performed in accordance with the NIH Guidelines for Care and Use of Laboratory Animals and the recommendations of the Association for Research in Vision and Ophthalmology. Mouse strain BALB/C was used throughout.
RNA extraction and RT-PCR
RNA from adult mouse eyes was isolated using RNAqueousTM-4PCR (Ambion, Inc., Austin, TX) or Trizol (Invitrogen Corp., Carlsbad, CA) per manufacturer's instructions. The RNA was treated with DNase I (Invitrogen) and RT-PCR was performed. Total RNA (1 μg) was reverse transcribed (Superscript II; Invitrogen) with random hexamers (Perkin Elmer, Boston, MA, or Promega, Madison, WI), and the resulting cDNA was used in the PCR reaction according to manufacturer's instructions (Platinum Pfx; Invitrogen). The following oligonucleotides were used: CCL27-ATG upstream: 5'-CCC GGG [GAA TTC] ATG ATG GAG GGG CTC TCC CCC-3' (1-21); CCL27/PESKY downstream: 5'-CCG CGG [GTC GAC] TTA GTT TTG CTG TTG GGG GTT-3'(673-693)
The following oligonucleotides were used to develop riboprobes for in situ hybridization; CCL27 intron 1, upstream: 5'-CCC GGG [GAA TTC] CAG GTA AGT TCT CCA G-3' (71-86); CCL27 intron 1, downstream: 5'-CCG GCC [GTC GAC] GAG AAC AAT AGG GCC-3' (197-211); CCL27 exon 3, upstream: 5'-CCC GGG [GAA TTC] GCT TCA CCT GGCTCG (537-551); PESKY exon 1', upstream: 5'-CCC GGG [GAA TTC] ATG TCT CCA ACA AGC CAG AGA CTA-3' (1-24); PESKY-exon 1', downstream: 5'-CCG GCC [GTC GAC] TTC TTG CTT CTG CTT AGT CTT GTT C-3' (69-93). The oligonucleotide position number is based on the A in the ATG site being +1. The PCR products generated were cloned into the EcoR I and Sal I sites of pBluescript II KS(+) vector (Stratagene, La Jolla, CA) for easy use of the T3 and T7 RNA polymerase sites for riboprobe production.
5' RACE was performed using the SMARTTM RACE cDNA Amplification Kit (BD Biosciences, San Jose, CA). The oligonucleotide used in the 5' RACE experiments was: CCL27 exon 2: 5'-CTC CTC AGC AGC CTG CTT GGG AGT GGC TGT C-3' (260-290); All 5' RACE products were cloned in TOPO TA vector (Invitrogen) and sequenced.
Real time PCR
RNA derived from whole eyes was subjected to real-time RT-PCR to determine the number of copies of the conventional CCL27 exon 1 and the alternative PESKY exon 1' per microgram of RNA. Primers used were as follows; CCL27 exon 1, upstream: 5'-ATG ATG GAG GGG CTC TCC CCC GCC AGC AG-3'; CCL27 exon 1, downstream: 5'-TTC AGG AGC CGG GCT CAG AAG CAA CAG-3'; PESKY exon 1', upstream: 5'-ATG TCT CCA ACA AGC CAG AGA CTA-3'; PESKY exon 1', downstream: 5'-TTG CTT CTG CTT AGT CTT GTT C-3'. Samples were mixed with 400 nM of each primer and 12.5 μl of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) reagent in a final volume of 25 μl. Real-time PCR was performed on the ABI Prism 7700 Sequence Detection System (Applied Biosystems), by heating samples at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Plasmids containing CCL27 exon 1 or PESKY exon 1' were also used to generate standard curves.
Eye collection and sectioning
BALB/C mice (2 months old) were euthanized by exposure to CO2. The eyes were removed immediately, frozen by immersion in isopentane (Sigma-Aldrich, St. Louis, MO) at -30 °C, and stored at -70 °C prior to sectioning. Consecutive serial sections of 20 μm thickness were cut in a cryostat at -16 °C, thaw mounted onto silanated slides (KD Medical, Columbia, MD) and stored at -70 °C until processed. A total of 100 eye sections obtained from 6 animals were processed for in situ hybridization.
In situ hybridization
Anti-sense and sense (control) riboprobes were labeled by in vitro transcription and in situ hybridization was performed as described earlier . Briefly, frozen sections were fixed for 10 min with a 4% paraformaldehyde solution in phosphate buffered saline (PBS), rinsed twice in PBS, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine HCl and dehydrated through a graded ethanol series. Each slide was treated with 150 μl of hybridization buffer containing 40,000 cpm/μl of 35S-labeled sense or anti-sense riboprobe. After hybridization for 16-18 h at 54 °C, sections were rinsed 4 times in 0.60 M NaCl and 0.06 M Na citrate, pH 7.0 (4X standard saline citrate buffer [SSC]) to remove coverslips and excess riboprobe. Non-hybridized riboprobes were digested by incubation with 40 μg/ml RNase A (Sigma-Aldrich) for 30 min at room temperature. After a final high stringency wash in 15 mM NaCl and 1.5 mM Na citrate, pH 7.0 (0.1X SSC) at 65 °C for 60 min, sections were dehydrated in graded ethanol containing 0.3 M ammonium acetate and air dried.
Sections were exposed to BioMax film (Eastman Kodak, Rochester, NY) along with 14C standards (Amersham Biosciences, Piscataway, NJ) for 4 and 7 days and developed in an automatic film developer X-OMAT (Eastman Kodak). mRNA expression was analyzed by measuring optical film densities on a G4 Macintosh computer using the public domain NIH Image 1.62 program.
Multiple CCL27 transcripts
RT-PCR amplification of total RNA from whole mouse eyes using oligonucleotides designed to amplify the full-length CCL27 chemokine mRNA yielded three products, one of which co-migrated with the product obtained from mouse skin, included as a positive control (Figure 1A). Sequence analysis of the PCR products identified them as splice variants of CCL27 (see schematic, Figure 1B). The largest product (AY744154) corresponded to a completely unspliced transcript retaining introns 1 and 2. The second largest transcript (AY744155) retained only intron 1. The smallest product was the fully spliced form of CCL27. Inclusion of intron 1 introduces a stop codon, which would truncate the open reading frame at 32 amino acids if translation begins at the usual CCL27 start site.
To search for other potential open reading frames that might be initiated upstream, we performed 5' RACE using an oligonucleotide specific for exon 2. Unexpectedly, when the 5' RACE products were cloned and 10 randomly selected clones were sequenced, 8 contained the PESKY first exon. Further examination of the sequence data of the 5' RACE products revealed two variants containing this alternative first exon (Figure 2A). One of these corresponds to the published PESKY sequence (AA271042). The other appears to be an alternatively spliced variant of a previously reported splice product (AK005398), with a unique 5' end. This alternative splice joins the PESKY first exon to genomic sequences about 2,000 bases upstream (Figure 2A).
Since the large number of PESKY clones among the 5' RACE products suggested that RNA transcripts containing the PESKY first exon are more abundant than those containing the chemokine CCL27 first exon, we measured the relative expression of these two alternative exons in whole eye RNA by real-time RT-PCR. The results showed that the PESKY first exon is approximately 4 fold more abundant than the CCL27 first exon in whole eye RNA (Figure 2B). RT-PCR on mouse eye RNA using oligonucleotides directed to the PESKY first exon (upstream) and exon 3 (downstream) yielded a single band (Figure 2C), demonstrating that RNA transcripts containing the PESKY first exon are not alternatively spliced downstream of exon 1.
To determine whether the observed CCL27 splice variants are differentially expressed in ocular tissues, we compared their expression in cornea, lens, and retina by RT-PCR. For this we used an upstream oligonucleotide directed either to the PESKY first exon or the canonical CCL27 first exon and a downstream oligonucleotide directed to exon 3 (Figure 3A). A single PESKY sequence was amplified from each of the tissues examined (Figure 3B), as in whole eye RNA (Figure 2C). However, the three splice products containing the CCL27 first exon were differentially expressed. Lens contained only the completely unspliced variant; retina contained predominantly the unspliced and partially spliced variants (with a trace of the fully spliced form); and cornea expressed all three forms, including the properly spliced CCL27 mRNA.
In situ hybridization
In situ hybridization was performed using 35S-labeled riboprobes complementary to the full length, fully spliced CCL27 mRNA (exons 1, 2, and 3), intron 1, exon 3, and the PESKY exon 1'. Results are shown for the riboprobe directed against the CCL27 mRNA (Figure 4B), which can hybridize to any transcript containing exons 1, 2, or 3. Specific hybridization signal was detected only in the retina (Figure 4B, arrows). Sections of the tail, used as positive controls, showed specific hybridization only in the skin (Figure 4E, arrows). Hybridization with riboprobes specific for intron 1, exon 3, and PESKY exon 1' also detected transcripts in the retina (not shown), as the RT-PCR results (Figure 3) would predict. We did not detect specific hybridization in lens or cornea with any of the riboprobes tested, although the more sensitive RT-PCR assay detected transcripts complementary to each of the riboprobes in these tissues(see Figure 3).
Analysis of the genomic sequence in and adjacent to the CCL27 locus reveals numerous potential splice donor and acceptor sites, making this region particularly favorable for splicing events. We have identified five distinct CCL27 derived splice variants that are expressed in the eye. Two of these (retaining either intron 1 only or introns 1 and 2) have not been reported previously, although a related EST is listed in GenBank (AW558992, UniSTS:178). This EST consists of exon 3 and part of intron 2 and may, thus, be derived from the variant that retains both introns 1 and 2. Intron retention is a common form of alternative splicing, found in up to 14% of all genes [10,11]. In the eye, retention of introns 1 and 2 showed distinct tissue specificity, with the fully spliced CCL27 mRNA expressed only in the cornea. Since the exon/intron boundaries of these CCL27 splice variants do not seem to be sub-optimal, the absence of the fully spliced form in retina and lens suggests that these tissues may lack certain critical splice factors. This possibility would be consistent with the previous observation that eye and retina are among the most common sites for expression of tissue specific splice forms .
All the ocular tissues examined also expressed CCL27 splice variants generated by alternative first exon usage, in which a previously reported upstream exon is substituted for the usual CCL27 first exon. This substitution generates a non-secreted protein, referred to as PESKY . Transcripts containing the PESKY first exon are expressed in lens, cornea, and retina, with the highest expression in the retina, as judged by in situ hybridization with a PESKY exon 1 specific riboprobe. We also detected a PESKY variant in the eye, which shows retention of an upstream intron. Interestingly, variants containing the PESKY first exon are the most abundant CCL27 derived transcripts in the eye, as judged by real time RT-PCR and random sequencing of 5' RACE clones. In view of this observation, our previous interpretation of data concerning CCL27 expression in normal and inflamed eyes  should be modified. The mRNA transcript designated 'CCL27' in that study was probably that of PESKY, a molecule that is neither skin specific nor a participant in inflammation. Indeed, in our previous study the level of CCL27 was unaffected by inflammation induced by TH1 cells, or the pathogenic process of experimental autoimmune uveitis .
The five CCL27 splice products expressed in the eye contain at least three open reading frames longer than 100 amino acids. As indicated above, the most abundant open reading frame corresponds to the non-secreted protein, PESKY . PESKY contains a nuclear localization signal and has been shown to accumulate in the nucleus of central nervous system neurons. Although little is known about PESKY function, a recent report indicates that it affects transcription of cytoskeletal genes in neuronal cells . The upstream intron which is included in the splice variant of PESKY found in the eye introduces an upstream ATG, generating a second open reading frame with 7 additional amino acids at the PESKY N-terminus. It is not yet clear whether this alternative form of the protein is indeed expressed.
The third CCL27 derived reading frame represented in the eye codes for the secreted chemokine, CCL27. This open reading frame is initiated in the conventional CCL27 first exon and extends through exons 2 and 3. Significantly, inclusion of intron 1 introduces a stop codon, which blocks expression of the chemokine in ocular tissues such as the retina and lens. In contrast, the cornea expresses the fully spliced form of CCL27 mRNA, and thus has the capacity to synthesize the secreted chemokine. Since this chemokine has been considered to be skin specific , its constitutive expression in the cornea is particularly noteworthy. Although we have not yet determined which corneal cell type expresses the CCL27 mRNA, corneal epithelial cells are likely candidates in view of their surface exposure, as well as the common embryological origin and structural similarity of corneal epithelium and skin. In the skin, CCL27 specifically targets CD4 lymphocytes that express the specific chemokine receptor CCR10 and the cell marker CLA. CD4 cells are known to play a critical role in corneal graft rejection and corneal inflammation during infection [13-16], although it is not yet clear whether the CD4 cells involved in these processes also express CCR10 and CLA.
The alternatively spliced CCL27 derived RNAs also have a number of short open reading frames which, if translated, would give rise to peptides of 30-50 amino acids. Several of these would be further shortened by cleavage of the signal peptide. Although RNAs with such short reading frames are generally considered to be non-coding, we can not rule out the possibility that some of these peptides may be expressed, possibly as biologically active products. Moreover, recent studies raise the possibility that the CCL27 splice variants may have regulatory roles as non-coding RNAs, affecting chromatin structure, the subcellular distribution of other RNAs, or the availability of proteins necessary for transcriptional and translational regulation [17,18].
We thank Dr. Esther Sternberg for advice and support throughout this project. This study was funded in part by an Intramural Research Award from the Integrative Neural Immune Program/Intramural Research Program on Women's Health, NIH, to PZ, IG, and LHT.
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