Injury-elicited differential transcriptional regulation of phospholipid growth factor receptors in the cornea

De-An Wang1, Haiming Du1, Jonathan H. Jaggar1, David N. Brindley2, Gabor J. Tigyi1, and Mitchell A. Watsky1

1 Department of Physiology, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163; and 2 Signal Transduction Research Group, Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The phospholipid growth factors (PLGFs), including lysophosphatidic acid (LPA), have been implicated in corneal wound healing. PLGF concentrations and activities are elevated after corneal injury. Using real-time PCR, we quantified receptor mRNA levels in the healing rabbit cornea. In intact corneas, transcripts for S1P1, LPA1, and LPA3 receptor subtypes were detected, as was lipid phosphate phosphatase 1 (LPP1). After wounding, the trend for endothelium and keratocytes was for significant decreases in transcript numbers for the three receptor subtypes, whereas epithelial cells showed increased transcript numbers, except for an S1P1 decrease in healing cells. LPP1 transcript numbers were decreased in keratocytes and endothelium, although LPP-specific activity was unchanged. LPA-elicited Ca2+ transients were significantly reduced in the healing endothelium. Consistent with reduced LPA3 receptor numbers, dioctylglycerol pyrophosphate, a selective antagonist, reduced LPA-induced Ca2+ transients 2.7-fold in nonwounded epithelium but only 1.5-fold in wound-healing endothelium. These data for the first time establish physiologically relevant differential changes in the expression of PLGF receptor subtypes and provide evidence for the changing role of LPA3 receptors in endothelial cells.

calcium; lysophosphatidic acid; phospholipid; wound healing; diacylglycerol pyrophosphate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LYSOPHOSPHATIDIC ACID (LPA) and sphingosine 1-phosphate (S1P) are lysophospholipid mediators with growth factor-like effects (12, 32) that have been implicated in wound healing in selected tissues including the cornea (2, 23, 31, 36). The extracellular actions of LPA and S1P are mediated by the endothelial differentiation gene family of G protein-coupled receptors. One cluster within this family, comprising the LPA1, LPA2, and LPA3 receptors, specifically recognizes LPA (3, 11), whereas the other cluster, consisting of S1P1, S1P2, S1P3, S1P4, and S1P5 receptors, is specifically activated by S1P (18). Each receptor has a distinct coupling pattern to various G proteins (15, 18, 28). Transient heterologous expression of LPA1/2/3 receptors results in the LPA-induced activation of intracellular Ca2+ transients (8, 9).

LPA and S1P facilitate wound closure in monolayers of endothelial cells isolated from adult bovine aorta and human umbilical vein (23). In vivo, LPA treatment stimulates wound closing and increases neoepithelial thickness in the skin (2). LPA accelerates intestinal epithelial wound healing by promoting intestinal epithelial cell migration and proliferation in vitro and in vivo (2, 31). In corneas, LPA stimulates proliferation in the three major cell types of the cornea (epithelial cells, keratocytes, and endothelial cells) in a dose-dependent manner (24, 36). This LPA-induced proliferative response is pertussis toxin sensitive in the corneal epithelial cells and keratocytes (36). Keratocytes from wounded corneas express an LPA-activated Cl- current that is also activated by an increase in cell volume and serum (35). LPA activation of the Cl- current in these cells is receptor mediated, and keratocytes isolated from nonwounded corneas do not express this current (24, 35). Biochemical examination of phospholipids present in the aqueous humor and lacrimal gland fluid from the rabbit eye has detected several phospholipid growth factors (PLGFs) including LPA, phosphatidic acid, alkenyl glycerophosphate, and lysophosphatidylserine (24). Injury to the cornea has resulted in an increased production of these PLGFs (24). All these data suggest that LPA, S1P, and their natural analogs might be involved in maintaining the integrity of the normal cornea and in promoting wound healing and/or cellular recovery after injury. This hypothesis presumes that PLGF responsiveness, determined by the expression of PLGF receptors, is maintained in the different cell types of the healing cornea.

The purpose of the present study was to investigate the expression of PLGF receptors in the three major cell types of the cornea and to assess wound healing-induced changes in their mRNA expression pattern. In addition, we determined the expression of mRNA encoding lipid phosphate phosphatase 1 (LPP1) as well as lipid phosphatase activity. LPP1 has been shown to degrade LPA at the cell surface (4, 22). Transcripts for S1P1, LPA1, and LPA3 receptors were detected in all three cell types, as was LPP1 mRNA. In endothelial cells and keratocytes, LPA and S1P receptor transcripts decreased during wound healing. In contrast, epithelial cell LPA1 and LPA3 expression increased during wound healing. In agreement with the decreased abundance of mRNA for the LPA3 receptor subtype in endothelial cells isolated from wound healing corneas, LPA-elicited Ca2+-transients were significantly diminished and lost their sensitivity to dioctylglycerol pyrophosphate (DGPP), an LPA3-selective antagonist. LPP1 message was decreased during wound healing in keratocytes and endothelial cells, although LPP1 activity was unchanged. These results support the hypothesis that PLGF receptors mediate the effects of these lipid mediators in corneal cells and that cell type-specific and differential transcriptional regulation of PLGF receptor expression might play a significant physiological role during corneal wound healing.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Rabbit corneal wounding and cell isolation. Corneas from anesthetized rabbits were freeze-wounded with the use of a liquid nitrogen-cooled brass probe by using a protocol described previously (35). At 44-72 h postwound, rabbits were killed, their eyes were enucleated, and the corneas were dissected from the globes. This was the time frame during which significant changes in LPA-activated Cl- current activity were found (35, 36). Buttons from injured areas were trephined to isolate the wound-healing zone. Cells from the trephined button, surrounding rim (the peri-wound area), and control corneas (no wounding) were isolated. Epithelial cells were scraped from the corneas, endothelium was peeled off with Descemet's membrane, and keratocytes were isolated by collagenase digestion of the stroma as described previously (35). All work was carried out in accordance with the Guiding Principles for Research Involving Animals and Human Beings.

RNA extraction and reverse transcriptase reaction. RT-PCR experiments utilized a total of 18 wounded corneas and 6 control corneas. Cells from the corneas were pooled, and total RNA was extracted from harvested cells using TRIzol (Life Technologies, Gaithersburg, MD). RNA was digested by using RNase-Free DNase I (2 U/µl; Stratagene, La Jolla, CA) in the presence of RNaseOUT (6 U/µl; Life Technologies) for 20 min at 37°C to eliminate genomic DNA contamination.

Total RNA (2 µg) was used as a template for cDNA synthesis by random primers using the ThermoScript RT-PCR system (Life Technologies) (33). The resulting cDNA was diluted 10-fold with diethyl pyrocarbonate-treated water (Sigma-Aldrich, St. Louis, MO) and was used as a template for standard and real-time PCR.

Primers were designed to highly conserved mammalian PLGF receptor cDNA sequences or to the rabbit sequences, where available (Table 1). Sequences of the rabbit LPA1 and S1P3 cDNA were kindly provided by Dr. James Rae (Mayo Clinic, Rochester, MN). Thirty-five PCR cycles, each consisting of 10 s at 94°C, 30 s at 55°C, and 1 min at 72°C, were performed. Amplification products were cloned into the pCRII-TOPO vector by using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) for sequencing. Primer compatibility with rabbit tissue was tested by using rabbit brain stem as a positive control.

                              
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Table 1.   Primers for RT-PCR and cloning

To monitor for contamination with genomic DNA, we also subjected all RNA samples to PCR (without reverse transcriptase treatment), and the resulting PCR products were analyzed on ethidium bromide-stained agarose gels. All samples subjected to DNase I digestion were found to be free of DNA contamination. To quantify receptor gene copy numbers in the samples, we developed standards using fragments of each of the receptors cloned into the pCRII-TOPO vector. Corresponding cRNAs were synthesized by using SP6 RNA polymerase with the mCAP mRNA capping kit (Stratagene). The cRNA concentration was measured with the GeneQuant RNA/DNA calculator (Pharmacia Biotech, Cambridge, UK). The yield of total RNA was measured by absorbance at 260 nm. The 260- to 280-nm (260/280) ratios of RNA samples were always >2.1 (7). The cRNA copy number was calculated according to the following equation: copy number = [(concentration × volume)/molecular weight] × 6.28 × 1023. The yield and quality of RNA were also checked by agarose gel electrophoresis.

Receptor cloning. Rabbit orthologs of several PLGF receptors, LPP1, and beta -actin were cloned to ensure appropriate design of real-time PCR primers. Mouse primers (Table 1) were used to amplify transcripts for LPA3, S1P1, LPP1, and beta -actin. LPA1 transcripts were amplified by using the sequence data provided by Dr. Rae. Products were subcloned into TOPO TA vector for sequencing.

Real-time PCR. Real-time PCR experiments utilized a total of 44 wounded corneas (72 h after wounding) and 10 control corneas. As with the RT-PCR experiments, cells were pooled before RNA extraction. PCR primers were designed using the Primer Express software (Table 2; Applied Biosystems, Foster City, CA). PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems). The SYBR Green PCR core reagents kit (Applied Biosystems) was used for the PCR reaction. The reaction contained 25 µl of SYBR reagent, 2 µl of diluted cDNA, and 30 nM primers in a 50-µl volume. The thermal cycling conditions involved an initial denaturation step at 95°C for 10 s and an extension step at 65°C for 1 min. Experiments were performed in triplicate for each data point. Quantitative values were obtained from the threshold cycle value (Ct), which is the point where a significant increase of fluorescence is first detected. The transcript number of rabbit beta -actin was quantified as an internal RNA control, and each sample was normalized on the basis of its beta -actin content. The relative PLGF receptor gene expression level of each sample was also normalized to the nonwounded control group (calibrator). Calculation of mRNA copy number was done by using the formulas available at http://dorakmt.tripod.com/genetics/realtime.html (17). Final results, expressed as N-fold difference in PLGF receptor expression relative to beta -actin and the nonwounded control, termed N, were calculated as N = 2(Delta Ct sample - Delta Ct calibrator), where Delta Ct values of the sample and calibrator were determined by subtracting the average Ct value of a PLGF receptor gene from the average Ct value of the beta -actin gene.

                              
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Table 2.   Primers for real-time PCR

Determination of lipid phosphate phosphatase activity. Cells from a total of 16 wounded (72 h after wounding) and 8 control rabbit corneas were examined. Cells were pooled from several corneas (separated as control, wound-healing buttons, and peri-wound rims) into individual tubes such that there were three individual groups for each category examined (n = 3 for each assay). Tissue samples were homogenized in a buffer solution containing (in mM, unless otherwise noted) 1% Nonidet, 10% glycerol, 50 HEPES, 137 NaCl, 1 MgCl2, 1 CaCl2, 10 Na2HPO4, 5 µg/ml aprotinin, 1 phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µM okadaic acid. Okadaic acid was employed to inhibit protein phosphatase activities because LPP activity is inhibited by vanadate and fluoride. Total LPP activity was measured by using [3H]palmitate-labeled phosphatidate (final concentration 0.6 mM) in the presence of 5 mM N-ethylmaleimide and 8 mM Triton X-100. The formation of [3H]diacylglycerol was determined after its purification with basic alumina (25).

Ratiometric intracellular Ca2+ measurements. Isolated corneal cells were incubated at room temperature for 20 min in NaCl Ringer's solution (in mM: 145 NaCl, 5 KCl, 2.5 CaCl2, 5 glucose, and 5 HEPES) containing the ratiometric fluorescent Ca2+ indicator fura 2-AM (2 µM) and 0.001% (wt/vol) Pluronic acid, during which time the cells adhered to a glass coverslip in a recording chamber. Cells were then washed for 10 min in bath solution to allow deesterification of the indicator dye. Cells were alternately excited at 340 or 380 nm using a PC-driven Hyperswitch (Ionoptix, Milton, MA). Background-corrected fura 2 ratios were collected at 510 nm every 0.2 s with the use of a photomultiplier tube and PC-based acquisition software (Ionoptix). Ca2+ fluorescence data were collected from single cells or groups of less than three attached cells. Intracellular Ca2+ concentrations ([Ca2+]i) were calculated by using the Grynkiewicz equation (16)
[Ca<SUP>2+</SUP>] = <IT>K</IT><SUB>d</SUB> <FR><NU>(R − R<SUB>min</SUB>) (S<SUB>f2</SUB>)</NU><DE>(R<SUB>max</SUB> − R) (S<SUB>b2</SUB>)</DE></FR>
where R is the 340/380 ratio; Rmin and Rmax are the minimum and maximum ratios determined in Ca2+-free and saturating Ca2+ solutions, respectively; Sf2/Sb2 is the Ca2+ free-to-Ca2+ replete ratio of emissions at 380-nm excitation; and Kd is the dissociation constant for fura 2. Rmin, Rmax, Sf2, and Sb2 were determined at the end of each experiment and in separate experiments by increasing the Ca2+ permeability of cells with ionomycin (10 µM) and perfusing cells with a high-Ca2+ (in mM: 140 KCl, 2.5 NaCl, 10 CaCl2, and 5 glucose) or Ca2+-free solution (in mM: 140 KCl, 2.5 NaCl, 5 glucose, and 1 EGTA). The in situ apparent dissociation constant (Kd) for fura 2 used in this study was 224 nM (16).

Data are expressed as resting [Ca2+]i or as the peak LPA-induced elevation in [Ca2+]i (i.e., LPA peak [Ca2+]i - baseline [Ca2+]i) for control and wound-healing cells. Inhibition of the LPA-induced intracellular Ca2+ transients by DGPP, the LPA3 receptor subtype-selective antagonist (8), was determined by the following experimental paradigm. Cells were first exposed to LPA (500 nM), followed by a 15-min washout in bath solution. DGPP (2 µM) was then applied for 15 min before a second application of LPA in the continued presence of DGPP. After a 15-min washout with bath solution, LPA (500 nM) was applied a second time to assess desensitization and/or rundown. The LPA-induced intracellular Ca2+ transient was calculated as the mean of the first and second application.

Statistical methods. Student's t-test was used for testing the null hypothesis that wounding elicited no statistically significant differences in mRNA abundance or baseline Ca2+ concentration, or that DGPP had no significant effect on the size of Ca2+ transients elicited by LPA. ANOVA was used to compare phosphatase activity levels between groups. P values <0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

S1P receptor transcripts in the corneal tissue. RT-PCR with the use of gene-specific primers based on the conserved transmembrane segments of the mouse S1P1, S1P2, S1P4, and S1P5 receptors (Table 1) detected only S1P1 transcripts in corneal endothelium, keratocytes, and epithelium (Fig. 1). The mouse S1P2, S1P4, and S1P5 receptor primers did amplify rabbit mRNAs encoding their respective receptor transcripts present in rabbit brain stem, indicating that the lack of product in these reactions was not due to inadequacy of the primers (data not shown). The rabbit-specific primer to the S1P3 receptor did not detect S1P3 receptor message in the corneal cell types (Fig. 1), although this primer readily amplified S1P3 transcripts present in the rabbit brain stem (data not shown). These results indicate that among the four S1P receptor subtypes, transcripts for the S1P1 receptor were the only ones ubiquitously expressed in all three corneal cell types.


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Fig. 1.   Phospholipid growth factor (PLGF) receptor subtypes in corneal epithelial cells (A), keratocytes (B), and endothelial cells (C). Total RNA was extracted from cells of control (lanes 1) and wounded rabbit corneas (lanes 2) and from the cells surrounding the wound (rim cells, lanes 3). RT-PCR was performed using the cDNA primers shown in Table 1. LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; M, molecular markers.

LPA receptor transcripts in the corneal tissue. Gene- and species-specific primers based on LPA1 receptors were used to assess the presence of this receptor subtype. LPA1 receptors were readily detectable in the three cell types of the cornea (Fig. 1). When primers based on the conserved putative transmembrane segments of the mouse orthologs of LPA2 and LPA3 receptors were used, amplification product was only detected for LPA3. When the same set of primers was used with mRNA isolated from rabbit brain stem, products were obtained that agreed in size with the expected size of the LPA receptor fragments (data not shown). The present results suggest that LPA1 and LPA3 receptors, but not LPA2 transcripts, are expressed in the cornea.

Receptor cloning. Rabbit beta -actin transcript was amplified, cloned, sequenced, and submitted to GenBank under the accession number AF404278. With the use of primers specific to rabbit beta -actin, transcripts were detected in every mRNA specimen, indicating that the quality of the RNA was adequate. Sequence alignment of the mouse and rabbit beta -actin orthologs showed a high degree of conservation (92%; data not shown).

The S1P1 receptor primer yielded a product in corneal endothelium, keratocytes, and epithelium that was cloned and sequenced. Sequences obtained from multiple clonal isolates yielded a single sequence corresponding to that of the rabbit ortholog of S1P1, which was submitted to GenBank under the accession number AF404275. Sequence alignment of the mouse and rabbit S1P1 receptor orthologs showed a high degree of conservation (89%; data not shown).

The primer set used to detect LPA1 and LPA3 receptors in corneal endothelium, keratocytes, and epithelium yielded two products that were cloned and sequenced. Sequence analysis showed two sequences obtained from multiple clonal isolates that represented the rabbit orthologs of the LPA1 and LPA3 receptors. The LPA3 sequence was deposited in GenBank under accession number AF404276. Sequence alignment of the mouse and rabbit LPA1 and LPA3 receptor orthologs also showed high degrees of conservation (92 and 83%, respectively; data not shown).

The LPP1 primers yielded a single product in all three corneal cell types that was also cloned and sequenced. This sequence represented the rabbit ortholog of LPP1 that was deposited in GenBank under accession number AF404277. Sequence alignment of the mouse and rabbit LPP1 receptor orthologs showed a high degree of conservation (86%; data not shown).

Real-time PCR. Quantitative real-time RT-PCR was utilized for the determination of mRNA copy numbers of the S1P1, LPA1 and LPA3 receptors and LPP1 enzyme, which were present in all three corneal cell types. Because beta -actin had unchanged mRNA levels in control, wounded, and peri-wound rim cells, it was chosen as an endogenous RNA control (Fig. 2).


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Fig. 2.   Quantitative changes of corneal PLGF receptor and beta -actin mRNAs measured using real-time PCR analysis. Total RNA from each of control, wound-healing (72 h), and peri-wound cells was analyzed. Known copy numbers of rabbit S1P1, LPA1, LPA3, and beta -actin cDNA were used as standards for determining absolute copy numbers. Values are means ± SE of 3 determinations. Scale on left corresponds to S1P1 and LPA1 transcript numbers; scale on right corresponds to LPA3 and beta -actin transcript numbers. *P < 0.001 vs. control. dagger P < 0.05 vs. control.

Epithelium. In the epithelium, S1P1 receptor transcripts were relatively low in abundance, constituting ~4,000 copies per 2 µg of RNA (Fig. 2). S1P1 receptors showed a decrease in the wound-healing area, whereas they were increased in the peri-wound rim. Both of these changes were statistically significant compared with the nonwounded epithelium. LPA1 receptors were the least abundant transcripts in the epithelium and showed a statistically significant approximately twofold increase in the wound-healing button and a modest but significant increase in abundance in the peri-wound rim. LPA3 receptor transcripts were the most abundant in the epithelium, with copy numbers in the range of 105 copies per 2 µg of RNA. During wound healing, the copy number for LPA3 receptors increased significantly almost threefold in the wound-healing button cells and twofold in the peri-wound rim.

Keratocytes. S1P1 receptor transcripts were also the least abundant in the keratocytes isolated from the stroma, with copy numbers comparable to those found in the epithelium (Fig. 2). The copy number of S1P1 decreased significantly in the wound-healing button, with no significant change in the rim compared with nonwounded keratocytes. LPA1 copy numbers were approximately fourfold higher in keratocytes compared with epithelium (Fig. 2). During wound healing, a statistically significant 3.5-fold decrease was detected in the copy number of LPA1 transcripts. In the rim, a twofold decrease was found, resulting in a copy number that was still significantly lower than in the nonwounded control but significantly higher than that in the wound-healing button. LPA3 transcripts were the most abundant PLGF receptor in keratocytes. The copy number of LPA3 decreased significantly in the wound-healing button and peri-wound rim. The abundance of LPA3 in the rim, although reduced relative to nonwounded control, remained significantly higher compared with the wound-healing button.

Endothelium. Endothelial cells contained the highest copy number of S1P1 receptor transcripts among the cells of the cornea, with copy numbers in the range of 104 copies per 2 µg of RNA (Fig. 2). The abundance of S1P1 transcripts decreased modestly but significantly during wound healing. Interestingly, S1P1 mRNA decreased substantially (~3.5-fold) in the endothelium of the peri-wound. LPA1 copy numbers detected in the endothelium were comparable to those seen in keratocytes. This receptor showed a modest decrease after wounding from 1.1 × 104 in nonwounded controls to 9.2 × 103 copies per 2 µg of RNA. As seen for S1P1, the expression of LPA1 transcripts was markedly reduced in the rim. LPA3 was the most abundant transcript detected in the endothelium, with copy numbers as high as ~3.5 × 105 per 2 µg of RNA. Expression of LPA3 transcript showed a >10-fold decrease in the wound-button and almost a 10-fold decrease in the endothelium of the peri-wound rim.

In summary, quantitative RT-PCR analysis revealed cell type- and receptor type-specific changes in the abundance of PLGF receptors mRNA transcripts for S1P1, LPA1 and LPA3 during wound healing in the cornea. LPA3 transcripts were found to be the most abundant in every corneal cell type studied, suggesting that this receptor could play a role in the maintenance of corneal integrity. In addition, the LPA3 receptor mRNA was downregulated in the inner two cell types of the cornea, whereas it was upregulated in the outer, epithelial cell layer. To seek a functional correlation between the downregulation of LPA receptor transcripts, we compared LPA-elicited Ca2+ responses in endothelial cells (see below).

LPP1 transcripts and lipid phosphate phosphatase activity. LPP1 transcripts were detected in all cell types of the cornea (Fig. 3A). Epithelial cells had the lowest LPP1 transcript numbers, which were not affected by wounding (Fig. 3B). Keratocytes and endothelial cells had higher LPP1 transcript copy numbers than epithelial cells, with the wounded region and rim having lower copy numbers than their control counterparts (although not significantly lower in keratocyte rim cells).


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Fig. 3.   RT-PCR and real-time PCR of lipid phosphate phosphatase 1 (LPP1) and lipid phosphate phosphatase activity in corneal cells. For PCR measurements, total RNA from each of control, wound-healing (72 h), and peri-wound cells was synthesized to cDNA. RT-PCR (A) was performed with the primers listed in Table 1, whereas real-time PCR (B) was performed using the primers listed in Table 2. Lipid phosphate phosphatase activity (C) was examined by measuring [3H]diacylglycerol formation from [3H]palmitate-labeled phosphatidate in control, wound-healing (72 h), and peri-wound cells. Values are means ± SE; n = 3 for each group. *P < 0.05 vs. control.

Keratocytes had the highest lipid phosphate phosphatase activity among the three cell types, which was 5.5- and 1.4-fold higher than that found in epithelial and endothelial cells, respectively (Fig. 3C). No significant changes were found in total lipid phosphate phosphatase activity during wound healing compared with nonwounded controls (Fig. 3C).

Changes in LPA-elicited Ca2+ responses in the healing endothelium. LPA evokes a transient elevation in [Ca2+]i in a variety of cell types. However, LPA regulation of intracellular Ca2+ has not been investigated in corneal cells. In addition, we sought to investigate the physiological consequences of changes in LPA receptor subtypes during wound healing on Ca2+ signaling in corneal cells. Endothelial cells were the only corneal cell type we found to respond to LPA with a change in [Ca2+]i (Fig. 4; epithelial cell and keratocyte data not shown).


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Fig. 4.   Ca2+ transients-induced by LPA in the epithelium. Endothelial cells from control and wounded corneas were loaded for 20 min with fura 2-AM. LPA (18:1; 500 nM) alone or together with 2 µM dioctylglycerol pyrophosphate (DGPP) was used to stimulate cells from control and wounded corneas. A: representative Ca2+ transients recorded from endothelial cells from a nonwounded control (top) and from a healing cornea (bottom). B: quantification of LPA-induced Ca2+ transients recorded in nonwounded control and wound-healing endothelial cells. Values are means ± SE; n = 4 and 5 for wounded and control, respectively. Note that the DGPP-sensitive component of the LPA response is decreased in the wounded epithelium.

[Ca2+]i- and LPA-induced intracellular Ca2+ transients were measured in control endothelial cells and endothelial cells isolated from wounded corneas (7 h after wounding). Resting [Ca2+]i in wound-healing endothelial cells (49 ± 24 nM; n = 5) was significantly lower than in nonwounded cells (113 ± 20 nM; n = 4; Fig. 4A). Consistent with the effects of LPA in other cell types, LPA at a concentration of 500 nM, which is a concentration ~10-fold higher than the Kd to the receptors, elicited a transient increase in [Ca2+]i with a mean peak size of 278 ± 65 nM (n = 4) in nonwounded cells (Fig. 4B). In contrast, the same concentration of LPA only elevated [Ca2+]i by 48 ± 13 nM (n = 5) in wound-healing cells. These results agree with the hypothesis that downregulation of LPA receptors could be responsible for the reduced LPA responsiveness of the endothelium.

To further substantiate the hypothesis that LPA receptor downregulation is responsible for the reduced Ca2+ response, we used DGPP, a selective antagonist of the LPA3 receptor with a Ki value of 104 nM. This is substantially lower than its Ki to LPA2, which is ~7 µM (8). LPA3 transcript numbers decreased by 80% in the wounded endothelium, whereas LPA2 transcript numbers decreased by only 10%; thus we hypothesized that the relative contribution of the LPA3 receptor to Ca2+ signaling should be the most significantly affected (reduced) in endothelial cells from healing corneas. In support of this hypothesis, 2 µM DGPP reduced the LPA-induced Ca2+ transient 2.7-fold in control cells but only 1.5-fold in the endothelium from the wound-healing button (Fig. 4). These data also suggest that the LPA3 receptor is responsible for most of the LPA-induced Ca2+ elevation in control cells.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

Several different growth factor receptors are involved in corneal wound healing, including receptors for platelet-derived growth factor, epidermal growth factor, transforming growth factor-beta , fibroblast growth factor, and hepatocyte growth factor (10, 21, 26, 27, 29, 37). We have shown the apparent induction of S1P2, S1P3, and LPA2 mRNA in tissue engineered from human corneas exposed briefly (5% for 3 min) to the detergent sodium dodecylsulfate (36). In that same study, we also found an apparent change of S1P1, S1P2, S1P3, LPA1, and LPA2 mRNA in human corneal epithelial cells following possible dedifferentiation of these cells after long-term culture (36). Because both of these experiments used in vitro systems and were performed with the use of a semiquantitative RT-PCR technique, the goal of the present study was to apply a quantitative method for the assessment of PLGF receptor mRNAs and to measure their wound-induced changes in the cornea in vivo. We found several differences in the receptor subtypes induced during wound healing in the rabbit cornea compared with our previous study with human cells. Not only is there a species difference, but we also used acutely dissociated corneal cells in the current study, which should be representative of the pattern of receptor expression in vivo, whereas we used cultured cells and immortalized cell lines obtained from cell and tissue culture in the previous study (36).

We have also shown that keratocytes acutely dissociated only from wounded rabbit corneas express a Cl- current that is activated by LPA via a receptor-mediated signaling pathway (35). Keratocytes from nonwounded corneas only rarely express this current. Part of the rationale for carrying out the present study was to determine whether the wound-induced Cl- conductance could be explained by LPA receptor mRNA induction during corneal wound healing. Our results are not consistent with this hypothesis and indicate that in keratocytes and endothelial cells the opposite occurs: the LPA receptor transcript number is decreased following wounding, at least at the time point we examined. This time point after wounding coincides with the maximum of the Cl- current activation in keratocytes. These results lead us to conclude that the Cl- channel activation must be the result of changes not directly related to the transcriptional regulation of LPA and S1P receptor mRNAs. Additional possibilities include posttranscriptional regulation of receptor activity and/or activation or amplification of signaling pathways leading from the remaining receptors to the Cl- channel.

We have used PCR primers based on mouse and rabbit sequences to detect the different PLGF receptor subtypes present in the three cell types of the rabbit cornea and have found a restricted expression pattern of only LPA1, LPA3, and S1P1 receptors. As part of this study, we cloned the rabbit orthologs of the LPA1, LPA3, S1P1 PLGF receptors as well as LPP1 and beta -actin, which was necessary for adequate real-time PCR primer design. These rabbit orthologs all showed an identity >80% with mouse sequences.

LPP1 decreases LPA responsiveness in different cell types (1, 20, 22, 39). For this reason, we have included LPP1 mRNA determination in our PCR experiments and have measured lipid phosphate phosphatase activity in the three different corneal cell types during wound healing. The present results show that the abundance of LPP1 transcript decreases after wounding in keratocytes and endothelial cells but not in epithelial cells. However, total lipid phosphate phosphatase activities were unchanged in all cell types during wound healing. This observation suggests that either the decrease in keratocyte and endothelial cell LPP1 mRNA levels is not associated with changes in activity or that other types of lipid phosphatases are also involved in the degradation of LPA in the tissues.

Little is known about the transcriptional regulation of PLGF receptors. Interestingly, S1P1 was originally isolated as a phorbol ester-inducible early response gene from vascular smooth muscle cells (19). Incubation of human blood T lymphocytes with mitogenic stimuli in vitro significantly alters the expression level of mRNA encoding PLGF receptors. These activation-mediated changes in T cell LPA1/2 receptor mRNA abundance correlate with major changes in LPA-mediated IL-2 production (14). Anti-Fas-, anti-CD2-, and anti-CD3 plus anti-CD28-induced apoptosis of T cells had no effect on the LPA1/2 or S1P2 mRNA abundance, whereas C6-ceramide substantially decreased LPA1/2 receptor mRNA abundance without affecting the S1P2 mRNA abundance (13). The present study provides the first comprehensive quantitative analysis of transcriptional regulation of PLGF receptors transcripts in tissues. Our findings are somewhat unexpected in that, for the most part, they show a decrease in PLGF receptor transcripts in the wound healing tissue. Moreover, we were surprised to find that, compared with S1P1, changes in LPA1 and LPA3 receptor transcripts were even more substantial in cells from the wound-healing corneas. Thus, in addition to S1P1, other PLGF receptor subtypes also show a transcriptional regulation induced by the physiological stimuli encountered during wound healing. LPA1 and LPA3 expression increased only in epithelial cells during wound healing. This is the opposite of what we detected in keratocytes and endothelial cells. In addition, we found significant changes in PLGF receptor transcript numbers in the peri-wound area, indicating that the entire tissue is affected by the wound healing process, not just the area directly affected by the wound. Thus our observations for the first time establish a cell type-specific regulation of LPA receptors within a tissue. While the physiological mechanism and inducers of LPA receptors in epithelial cells remain unknown at the present time, different trends of mRNA regulation take place in corneal keratocytes and endothelial cells. In the control cornea, the epithelium is exposed to tears and the external environment, while the barrier created by the tight junctions between these cells limits keratocytes and endothelial cells to exposure to factors only within the anterior segment of the eye. After wounding, however, the epithelial barrier is disrupted and all cell types are exposed to tears and external factors, although this exposure ends as soon as the epithelial barrier is restored. We speculate that factors regulating the expression of the LPA receptor genes are predominant in the tear and aqueous humor, because these are the biological fluids that bathe the epithelial and endothelial layers. Our earlier report provided evidence for the increased generation of LPA and its analogs in postinjury lacrimal gland fluid and aqueous humor. The transcriptional upregulation of LPA receptors together with the increased production of the ligand following injury further supports our hypothesis of a role for these factors during corneal epithelial wound healing.

LPA-induced changes in [Ca2+]i were only observed in the corneal endothelial cells. Previous work has found that corneal endothelium intracellular Ca2+ can be increased through receptor-mediated pathways by histamine (5, 6), purinergic agonists (5, 30), bradykinin (5, 40), and endothelin-1 (5, 38). Adrenergic agonists decreased [Ca2+]i (34). None of these studies were performed in rabbit cells. The current study clearly shows that LPA can be added to this list and is the first agonist shown to elevate intracellular Ca2+ levels in rabbit corneal endothelial cells. Moreover, we detected a correlation between the decrease in LPA3 receptor mRNA and the changes in the DGPP-sensitive component of the Ca2+ response in endothelial cells. Clearly, more experiments are necessary to exclude changes in the coupling efficiency of the signaling pathway linking LPA receptors to the mobilization of intracellular Ca2+. Nevertheless, our data establish a distinct change in the pharmacological properties of LPA receptors regulating Ca2+ homeostasis in the endothelium during wound healing.

Altogether, the present study for the first time provides evidence that physiological conditions occurring during wound healing affect the expression of PLGF receptors mRNA and that these changes are accompanied by differences in the pharmacology of LPA-induced Ca2+ responses. These results open many new questions regarding the mechanisms underlying these changes, which must be addressed in future studies.


    ACKNOWLEDGEMENTS

We thank J. Dewald for help in performing the LPP assays.


    FOOTNOTES

This work was supported by National Institutes of Health grants EY-12821 (to M. A. Watsky) and HL-61469 (to G. J. Tigyi), National Science Foundation Grant IBN-9728147 (to G. J. Tigyi), and Canadian Institutes of Health Research Grant MT10504 (to D. N. Brindley).

Address for reprint requests and other correspondence: M. A. Watsky, Dept. of Physiology, Univ. of Tennessee Health Sciences Center, 894 Union Ave., Memphis, TN 38163 (E-mail: mwatsky{at}physio1.utmem.edu).

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

August 14, 2002;10.1152/ajpcell.00323.2002

Received 11 July 2002; accepted in final form 13 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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