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J Biol Chem, Vol. 275, Issue 19, 14573-14578, May 12, 2000

Gelsolin Binding and Cellular Presentation of Lysophosphatidic Acid^*

Edward J. Goetzl, Hsinyu Lee, Toshifumi Azuma^§, Thomas P. Stossel^§, Christoph W. Turck, and Joel S. Karliner^¶

From the Departments of Medicine and Microbiology-Immunology and ^¶ Veterans Affairs Medical Center, University of California, San Francisco, California 94143-0711 and ^§ Department of Medicine, Hematology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lysophosphatidic acid (LPA) in biological fluids binds to serum albumin and other proteins that enhance its effects on cellular functions. The actin-severing protein gelsolin binds LPA with an affinity (K_d = 6 nM) similar to that of the G protein-coupled LPA receptors encoded by endothelial differentiation genes 2, 4, and 7 (Edg-2, -4, and -7 receptors) and greater than that of serum albumin (K_d = 360 nM). At concentrations of 10% or less of that in plasma, which are observed in fluids of injured tissues, purified and recombinant gelsolin augment LPA stimulation of nuclear signals and protein synthesis in rat cardiac myocytes (RCMs) that express Edg-2 and -4 receptors. At concentrations of 20% or more of that in plasma, gelsolin suppresses LPA stimulation of RCMs. The lack of effect of gelsolin on RCM responses to monoclonal anti-Edg-4 receptor antibody plus a phorbol ester without LPA attests to its specificity for LPA delivery and the absence of post-receptor effects. Inhibition of gelsolin binding and cellular delivery of LPA by L--phosphatidylinositol-4,5-bisphosphate (PIP2) and peptides constituting the two PIP2 binding domains of gelsolin suggests competition between LPA and PIP2 for the same sites. Thus, delivery of LPA to RCMs is affinity-coupled to Edg receptors by gelsolin in a PIP2-regulated process.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lysophosphatidic acid (LPA)¹ and sphingosine 1-phosphate (S1P) are highly active cellular growth factors that are generated enzymatically from cellular membrane precursors and secreted by activated platelets, leukocytes, epithelial cells, and some types of tumors (1-3). Extracellular LPA and S1P are bound extensively by serum albumin, which also provides for their delivery to cellular receptors (4-6). In one of the earliest studies designed to identify cellular receptors for LPA, the extension of a receptor affinity-labeling method to serum proteins in one experiment detected interactions of LPA with proteins of 15, 28, and 80 or more kDa as well as serum albumin (7). However, these proteins were not resolved or characterized further, and no studies ever were conducted of equilibrium binding of LPA to serum albumin or any other protein.

Extensive investigations have determined the fatty acid and phospholipid binding properties of serum albumin, which include equilibrium dissociation constants as low as 10 nM for some fatty acids and as high as 300 to 1,000 nM for larger and more polar fatty acids with limited access to the high affinity pockets (8-10). However, these studies using classical techniques did not include analyses of binding of LPA to serum albumin, in part due to the high levels of unspecific binding attributable to the amphipathic nature of LPA. It was discovered recently that one or more proteins secreted into serum-free medium of cultured rat cardiac myocytes (RCMs) delivered LPA, but not S1P, to RCMs and some other cells more effectively than fatty acid-free bovine serum albumin (faf-BSA) (11). LPA regulates the actin-binding and -severing activities of gelsolin, an approximately 85-kDa cytosolic constituent of myocytes and a myocyte-derived plasma protein, by mechanisms similar to those of L--phosphatidylinositol-4,5-bisphosphate (PIP2), which binds to two distinct sites of gelsolin (12-14). This raised the possibility that gelsolin, which normally is present in plasma and extracellular fluids at a concentration of 100 to 250 µg/ml, may be one of the RCM-derived proteins capable of binding LPA and then either sequestering it or delivering it to cellular receptors. A sub-family of G protein-coupled receptors encoded by endothelial differentiation genes (EdgRs) recently has been shown to bind and transduce signals from several lysolipid phosphate mediators (15-22). At nanomolar concentrations of ligands, Edg-2, -4, and -7 receptors are specific for LPA and Edg-1, -3, and -5 receptors for S1P.

This study was designed to quantify equilibrium binding of LPA to gelsolin and faf-BSA, characterize the gelsolin binding sites for LPA in relation to those for PIP2, and investigate the capacity of gelsolin to deliver LPA to RCMs and elicit RCM nuclear, biochemical, and functional responses.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemical Reagents and Antibodies-- Sources of chemicals were as follows. Purified human plasma gelsolin was from Sigma, human recombinant gelsolin was from Biogen Corp., Cambridge, MA, recombinant Clostridium botulinum ADP-ribosyltransferase (C3 exoenzyme), was from List Biological Laboratories, Inc., Campbell, CA, pRL-CMV Renilla luciferase vector was from Promega, Inc., Madison, WI, and FuGENE 6 transfection reagent was from Roche Molecular Biochemicals Corp. Recombinant gelsolin has the proper pairing of sulfhydryl groups to ensure native conformation and full biological activity, as described previously (23, 24) and reproven recently (Biogen Corp.). The villin 133-YDVQRLLHVKGKRNV-147 (25), gelsolin 135-KSGLKYKKGGVASGF-149 (13) (gel P1), gelsolin 150-KHVVPNEVVVQRLFQVKGRR-169 (14) (gel P2), and control random gelsolin 150-VNPEVLRVFKQHVVRGVRKQ-169 substituent peptides were synthesized using a solid-phase system (Model 433 Peptide Synthesizer, Perkin-Elmer) with fluorenylmethoxycarbonyl-protected amino acids (Bachem, Inc., Torrance, CA), purified by high performance liquid chromatography, and verified by mass spectrometry with an LCQ ion trap (Finnigan MAT, San Jose, CA). Mouse monoclonal antibodies against substituent peptides of human Edg-3 (amino acids 1-21), Edg-4 (amino acids 9-27), and Edg-5 (amino acids 303-322) with high interspecies homology were purified and used for Western blots at 0.1-0.3 µg/ml (26-28) (Antibody Solutions, Palo Alto, CA). A rabbit polyclonal antiserum to rodent and human Edg-2 was kindly provided by Dr. Jerold Chun (University of California San Diego). The cross-reactivity of each antibody with heterologous Edg proteins was less than 1%. Mouse monoclonal anti-human gelsolin antibody was purchased from Transduction Laboratories (Lexington, KY).

Cell Culture and Quantification of Cellular Protein Synthesis-- Rat cardiac myocytes (RCMs) of 90% purity with up to 10% fibroblasts from 1-day-old Harlan Sprague-Dawley rat pups were cultured in Dulbecco's modified minimal Eagle's medium with penicillin and streptomycin (DMEM⁺) and 10% fetal bovine serum at 37 °C in 5% CO₂ (29, 30). Replicate layers of RCM were preincubated with pertussis toxin for 6 h and C3 exoenzyme for 30 h. RCMs were serum-deprived 24 h before each study in DMEM⁺ or DMEM⁺ containing 1-100 µg/ml faf-BSA or 0.1-100 µg/ml of gelsolin, and the medium was replaced just before RCM stimulation at the beginning of a study. To quantify incorporation of [³H]leucine into proteins by RCM, replicate monolayers of 3-4 × 10³ RCM were serum-deprived and cultured in 12-well plates for 72 h with removal of medium and readdition of antagonists and stimuli in fresh medium every 24 h. In these and all other studies, LPA was added in an aliquot of 100 µg/ml faf-BSA equal to 1/50 the volume of medium in each well. This prevents LPA binding to non-cellular surfaces even at aqueous concentrations far below the critical micellar level of approximately 1 mM (5). One µCi of [³H]leucine was added to wells, and incubation was continued for 12 h. RCM then were washed twice with 1 ml of phosphate-buffered saline, fixed in 0.5 ml/well of trichloroacetic acid at 5 g/100 ml of distilled water, held at room temperature for 15 min, and solubilized by incubation at 37 °C for 2 h in 0.2 ml of 5 g % sodium dodecyl sulfate for determination of radioactivity.

Western Blots-- Replicate suspensions of 1 × 10⁷ RCMs, fibroblasts from preparations of RCMs, and HTC4 cell transfectants were washed three times with 10 ml of ice-cold Ca²⁺- and Mg²⁺-free phosphate-buffered saline, resuspended in 0.3 ml of cold 10 mM Tris-HCl (pH 7.4) containing a protease inhibitor mixture (Sigma), 0.12 M sucrose, and 5% glycerol (v:v), and proteins were extracted (27). In initial analytical gels, aliquots of supernatant containing 1-100 µg of protein were electrophoresed, transferred, and immunostained (27).

Quantification and Analysis of Gelsolin and Serum Albumin Binding of LPA-- Two polished lucite 8-cm diameter × 12-mm thickness disks with eight 10-mm diameter half-wells in each held a 10-kDa pore cut-off middle layer of acetylated dialysis membrane between the half-wells of each chamber. One half-well received 200 µl of either 5 µM faf-BSA or 2 µM gelsolin in phosphate-buffered saline with Ca²⁺ and Mg²⁺, and the other received 200 µl of [³H]LPA (150,000 dpm or 800,000 dpm in competition studies) in Ca²⁺- and Mg²⁺-containing phosphate-buffered saline with 20 µg/ml (0.3 µM) faf-BSA. Non-radioactive LPA at 0.1 nM-1 µM or a range of concentrations of binding inhibitors were added at equal concentrations to both half-wells of some sets. After 48 h of incubation with rolling at 4 °C, replicate aliquots of 50 µl were counted to determine the difference between dpm in a protein-containing half-well and in the non-protein half-well of each compartment. After normalization for recovery, which ranged from 49% to 70%, each difference was expressed as a percentage of dpm in the reference set lacking any inhibitor (100%). Plots of % of control binding versus LPA concentration were used to derive K_d values by the MacLigand program of Dr. Robert E. Williams (UCLA) and for corroboration by the LIGAND program of Biosoft (Cambridge, MA). Plots of % of control binding versus inhibitor concentration permitted calculation of IC₅₀ values. Binding data were analyzed for statistical significance by one-way analysis of variance and Fisher's protected least significant difference (StatView, Abacus Concepts, Berkeley, CA).

Transfections and SRE Reporter Assay-- Replicate monolayers of 0.3-1 × 10⁵ RCMs in 12-well plates of DMEM⁺ were lipotransfected with 100 ng/well of an SRE firefly luciferase reporter plasmid (16) and 5 ng/well of pRL-CMV Renilla luciferase vector (Promega) using FuGENE 6. After 24 to 30 h, the RCM were washed and covered with 1 ml of DMEM⁺ before stimulation by LPA or a combination of monoclonal anti-Edg-4 antibody and phorbol myristate acetate (PMA). Each firefly luciferase luminometric value was corrected for process differences by division with the Renilla luciferase luminometric signal from the same well (31).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Direct nuclear signaling by LPA recruits the transcription proteins serum response factor and ternary complex factor to form a ternary complex with the SRE in promoters of numerous immediate-early growth-related genes. RCM transfected with an SRE luciferase reporter, permitting quantification of direct nuclear signaling, responded modestly only to 10⁶ M LPA in protein-free medium, only to 10⁶ M LPA in 1 and 10 µg/ml faf-BSA, and to 10⁷ M and 10⁶ M LPA in 100 µg/ml faf-BSA (Fig. 1). In contrast, delivery of LPA to RCM by 1, 3, and 10 µg/ml gelsolin resulted in significant responses at 10⁹-10⁶ M LPA, with maximal mean increases in signal of 3- to 4-fold. The effectiveness of purified gelsolin was steeply dependent on concentration, with no responses at 0.3 µg/ml, maximum signals at 1-10 µg/ml, and lower than maximum responses at 30 µg/ml (Fig. 1). In two studies that included 100 µg/ml purified gelsolin, SRE luciferase signals were not significantly higher or lower than medium control values at 10⁸-10⁶ M LPA. In limited studies, 10⁸ M and 10⁷ M LPA delivered by 3 µg/ml human recombinant gelsolin elicited respective mean increases (±S.D.) in SRE luciferase signals to 312 ± 48% and 274 ± 60% of control (both p < 0.01). The presentation of LPA to RCMs by gelsolin thus attained a peak at concentrations lower than 10% that in plasma and decreased at levels 20% or more of that in plasma. In contrast, gelsolin did not enhance S1P-evoked SRE luciferase responses in parallel experiments (data not shown). Furthermore, neither purified or recombinant gelsolin nor faf-BSA alone evoked SRE luciferase responses (Fig. 1).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   SRE luciferase reporter assay of nuclear signaling of rat cardiac myocytes by LPA. Data and each error bar depict the mean ± S.D. of the results of three or four different studies. The medium alone control values were 2906, 3130, 8020, and 10,402 luminometer units. Human purified plasma gelsolin was used for the studies presented in this figure. The levels of significance of increases above medium controls were determined with a paired sample Student's t test. #, p = 0.05; +, p < 0.05; *, p < 0.01.

As RCM do not proliferate in culture, when assessed by increases in cell counts, the functional consequences of their activation by LPA were investigated by measuring enhancement of protein synthesis characteristic of myocyte hypertrophy, as quantified by uptake of [³H]leucine into trichloroacetic acid-precipitated cellular proteins. The level of protein synthesis by RCM was increased by LPA delivered by purified plasma gelsolin (Fig. 2). The increases in incorporation of [³H]leucine were significant at 10⁸-10⁶ M LPA with 3 µg/ml and 10 µg/ml gelsolin and attained a peak at 10⁷ M and 10⁶ M with 3 µg/ml and 10⁸ M and 10⁷ M with 10 µg/ml gelsolin. In limited studies, 10⁸ M and 10⁷ M LPA delivered by 3 µg/ml human recombinant gelsolin elicited respective mean increases (±S.D.) in incorporation of [³H]leucine to 194 ± 44% and 236 ± 18% (p < 0.05 and <0.01, respectively) of control. There was a marginally significant increase in RCM protein synthesis with 10⁶ M LPA delivered by 100 µg/ml faf-BSA. In contrast, LPA delivered by 1 µg/ml gelsolin or by 1 µg/ml or 10 µg/ml faf-BSA had no effect on RCM protein synthesis (Fig. 2). The maximal responses to gelsolin-LPA far exceeded in magnitude those evoked by faf-BSA-LPA. Neither gelsolin nor faf-BSA alone altered RCM protein synthesis.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Stimulation by LPA of increases in incorporation of [3H]leucine into proteins by rat cardiac myocytes: purified gelsolin and LPA concentration dependence relationships. Data and each error bar depict the mean ± S.D. of the results of three different studies. The medium alone control values (100%) were 24,902, 18,655, and 29,342 dpm. The levels of significance of changes from control levels were determined with a paired sample Student's t test. #, p = 0.05; +, p < 0.05; *, p < 0.01.

The identity of gelsolin as the protein delivering LPA to RCM was confirmed by the suppression of SRE luciferase responses with a neutralizing mouse monoclonal antibody to human gelsolin. RCM SRE luciferase responses similar in magnitude to those described in Fig. 1 were again evoked when LPA was delivered by gelsolin or faf-BSA (Fig. 3). In contrast, neither gelsolin alone or gelsolin with the monoclonal anti-gelsolin antibody increased SRE luciferase reports. The responses elicited by gelsolin-LPA, but not faf-BSA-LPA, were suppressed significantly by means of more than 70% with mouse anti-gelsolin antibody (Fig. 3).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Inhibition by monoclonal anti-gelsolin antibody of delivery of LPA to rat cardiac myocytes by human purified gelsolin. Data and each error bar depict the mean ± S.D. of the results of three different studies. The concentration of anti-gelsolin antibody and isotype control IgG was 10 µg/ml. The levels of significance of inhibition of SRE luciferase responses were determined with a paired sample Student's t test. #, p = 0.05; +, p < 0.05; *, p < 0.01. The number in parentheses above each bar showing significant suppression of responses is the mean percentage suppression of the corresponding response with control IgG.

Monoclonal antibodies to the EdgRs that bind and transduce signals from LPA recently were shown to elicit growth-related responses of some cells when introduced with PMA (32). Thus the EdgRs of RCM were analyzed immunochemically first and then for their capacity to transduce antibody-induced signals to determine if gelsolin influenced any receptor or post-receptor elements of RCM responses in addition to LPA delivery. RCM expression of Edg-2 and -4 receptors for LPA and Edg-3 and -5 receptors for S1P was examined in Western blots developed with the monoclonal antibodies to human and rodent Edg-3, -4, and -5 receptors and rabbit IgG polyclonal antibodies to human and rodent Edg-2Rs with stimulatory potential (Fig. 4). One predominant protein antigen was observed with each anti-EdgR antibody in extracts of RCM that was the same size as the protein detected by that antibody in extracts of HTC4 rat hepatoma cell transfectants bearing the corresponding recombinant EdgR (21, 31). The apparent quantity of each EdgR antigen extracted from purified cardiac fibroblasts, which represent up to 10% of the cells in RCM preparations, was lower than that in RCMs despite the application of 2.5 times more protein from the fibroblasts (Fig. 4). RCMs thus appear to be the principal cells in fresh preparations that express EdgRs and can respond by EdgR-dependent mechanisms both to LPA and anti-EdgR antibodies combined with PMA. Two toxins active against transductional components required for EdgR signaling were employed to determine if RCM EdgRs signal by the expected mechanisms. Both pertussis toxin and C3 exoenzyme separately significantly suppressed the increases in SRE luciferase activity induced by LPA delivered to RCM by either 100 µg/ml faf-BSA or 3 µg/ml gelsolin (Table I). Application of pertussis toxin and C3 exoenzyme together inhibited SRE luciferase reporter responses to 10⁷ M LPA with 3 µg/ml gelsolin by a mean of 83% (n = 2).

View larger version (57K): [in this window] [in a new window]   Fig. 4.   Western blot analyses of the expression of Edg-2, -3, -4, and -5 receptors by rat cardiac myocytes and fibroblasts. The three samples are 2 µg of protein from HTC4 rat hepatoma cells (T), which were stably transfected with the respective EdgRs, 10 µg of protein from rat cardiac fibroblasts (F), which constitute up to 10% of cells in cardiac myocyte preparations, and 4 µg of protein from rat cardiac myocytes of 90% purity (M). No Edg-3, -4, or -5 receptor antigen was detected in untransfected HTC4 cells with monoclonal antibodies, but a low level of Edg-2R antigen was revealed by the rabbit anti-mouse Edg-2R antibodies. The marginal lines show the positions of the 45-kDa and 66-kDa protein molecular mass standards for Edg-2 and -4 and the 45 kDa molecular mass standard for Edg-3 and -5.

As Edg-4R was one of the most prominent LPA receptors of RCMs and monoclonal anti-Edg-4R antibody with PMA has evoked SRE luciferase responses in other cells (32), concentrations of anti-Edg-4R antibody and PMA, which alone did not elicit signals, were applied to RCM in several combinations (Table II). These combinations of monoclonal anti-Edg-4R antibody and PMA evoked mean SRE luciferase responses of RCM from 194% to 235%, which were similar in magnitude to those induced by LPA with gelsolin. However, gelsolin did not significantly modify the signals attained by anti-Edg-4R antibody-based stimulation (Table II). Thus gelsolin does not appear to have major effects on post-EdgR cellular elements of signaling of RCMs.

The capacity of concentrations of gelsolin lower than 10% that of normal plasma to deliver LPA to RCMs with optimal efficiency suggested high affinity binding of LPA to gelsolin. The results of analyses of equilibrium binding of [³H]LPA to gelsolin and faf-BSA showed greater displacement from the former by all concentrations of non-radioactive LPA but especially large differences between gelsolin and faf-BSA at 3 × 10⁸ M to 3 × 10⁷ M (Fig. 5). Both recombinant and purified human gelsolin demonstrated similarly greater displacement of [³H]LPA at low LPA concentrations. Purified and recombinant gelsolin had respective mean K_d values ± S.E. (n = 3) of 6.2 ± 2.5 nM and 32 ± 7.8 nM, as contrasted with 357 ± 64 nM for faf-BSA. Assessment of valences of the binding proteins indicated a range of 1.2 to 1.8 for gelsolin and 3.2 to 5.6 for faf-BSA. S1P and lysophosphatidylcholine at 10⁶ M both failed to displace [³H]LPA from gelsolin.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   LPA concentration dependence of displacement of [3H]LPA bound to purified human plasma gelsolin (G pur.), human recombinant gelsolin (G rec.) and fatty acid-free bovine serum albumin (S). Each point is the mean ± S.E. of the results of two different studies carried out in duplicate. The mean net specific binding of 150,000 dpm of [3H]LPA to 5 µM faf-BSA was 52,822 and 56,582 dpm, to 2 µM plasma gelsolin was 9,740 and 10,851 dpm, and to 2 µM recombinant gelsolin was 20,412 and 20,390 dpm in the two studies in the absence of non-radioactive LPA (100%).

A relationship between gelsolin binding of LPA and of PIP2 was examined by PIP2 competitive inhibition of both gelsolin binding of [³H]LPA and cellular presentation of LPA by gelsolin. A 300-fold molar excess of PIP2 inhibited LPA binding to gelsolin by a mean of 31% (Fig. 6A), and a 100-fold molar excess of PIP2 inhibited LPA presentation to RCM by a mean of 56% for nuclear signaling (Fig. 6B) and 38% for protein synthesis (Fig. 6C). In contrast, there was no significant inhibition of LPA binding to faf-BSA or of faf-BSA enhancement of LPA effects on cardiomyocytes by PIP2 (data not shown). Substituent peptides of gelsolin (gel P1 and gel P2), each of which represents one of the two PIP2-binding sites of intact gelsolin and of another actin-binding protein termed villin (villin P), were used as gelsolin competitive inhibitors at concentrations 3-300-fold higher than 0.03-0.3 µM intact gelsolin (13, 14, 25). Gel P1 and gel P2 significantly suppressed binding of [³H]LPA to 0.3 µM intact gelsolin at 3-fold and 30-fold higher molar concentrations (Fig. 6A). Gel P1 and gel P2 significantly suppressed signaling of SRE luciferase by LPA-0.03 µM gelsolin at 30-fold and 300-fold higher molar concentrations (Fig. 6B). The two gelsolin peptide sites of PIP2 binding also significantly inhibited induction of RCM protein synthesis by LPA with 0.03 µM gelsolin at a 300-fold higher molar concentration (Fig. 6C). In contrast, a control nonsense sequence gelsolin peptide 2 had no effect on intact gelsolin binding or delivery of LPA (Fig. 6). Villin P, which is structurally homologous to gelsolin PIP2 binding peptides, also inhibited gelsolin binding and cellular presentation of LPA at similar molar concentration ratios with intact gelsolin. None of the gelsolin inhibitory peptides significantly altered faf-BSA binding or delivery of LPA.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Competitive inhibition by PIP2 and binding site peptides of actin-binding proteins of [3H]LPA binding to human purified gelsolin (A) and of LPA-gelsolin stimulation of SRE luciferase (B) and incorporation of [3H]leucine into proteins (C) in rat cardiac myocytes. A, inhibition of binding of 800,000 dpm of [3H]LPA to 0.3 µM human purified plasma gelsolin. Data and each error bar depict the mean ± S.E. of the results of three or four different studies, except for gelsolin peptide 1 and control gelsolin peptide 2, which are the mean of duplicate results. The control levels of binding of [3H]LPA alone (0% inhibition) ranged from 6,802 to 20,425 dpm. B, inhibition of stimulation of SRE luciferase activity by 107 M LPA, 3 µg/ml gelsolin. Data and each error bar depict the mean ± S.D. of the results of three different studies. The control levels of SRE luciferase with LPA and gelsolin alone (0% inhibition) were 9,332, 3,241, and 17,089 luminometer units. C, inhibition of stimulation of incorporation of [3H]leucine into proteins by LPA and gelsolin. Data and each error bar depict the mean ± S.D. of the results of three different studies. The 3 µg/ml gelsolin and 107 M LPA alone control values (0% inhibition) were 93,629, 84,710, and 81,505 dpm. The levels of significance of mean percentage inhibition in A, B, and C were determined with a paired sample Student's t test. #, p = 0.05; +, p < 0.05; *, p < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The expression of one or more EdgRs by all but a few types of mammalian cells has suggested that the activities of LPA and S1P are regulated principally by alterations in their effective local concentrations. The relative roles of production, non-receptor protein binding, and biodegradation in the determination of concentrations of LPA at cell-surface EdgRs have not been elucidated in physiological settings. The amount of LPA generated is a function of membrane perturbation of cellular sources and recruitment of a sequence of phospholipases, which are induced by diverse stimuli (1-3). Many lysophospholipases with different cellular localization, substrate specificity, and susceptibility to stimulation and inhibition may degrade or otherwise inactivate LPA (33). Thus, LPA is produced and biodegraded at sites of tissue reactions in the course of many physiological responses and pathological processes. Less is known of the mechanisms for stabilization of LPA in tissues and specific cellular delivery of LPA.

Plasma gelsolin, which is derived principally from myocytes (14, 34), binds LPA with an affinity similar to that of EdgRs and, exceeding, that of serum albumin or other known plasma proteins (Fig. 5). The identity of gelsolin as the LPA-binding protein of purified plasma preparations was verified by the similar activity of recombinant gelsolin (Fig. 5) and the neutralizing effect of monoclonal anti-gelsolin antibody (Fig. 3). This implies that LPA derived from any primary cellular source first will bind with low affinity to the much greater density of serum albumin sites and then may be captured effectively by high affinity binding to gelsolin. The present data suggest that gelsolin could serve two functions in regulating expression of LPA activity. Gelsolin concentrations in plasma normally are 150 to 250 µg/ml and rarely decrease to lower than 60 µg/ml even in cases of extensive trauma, where there is extreme dilution and actin-trapping (35-39). At such high plasma concentrations, therefore, the first function of gelsolin may be as a high affinity plasma carrier, which protects a portion of circulating LPA from biodegradation, prevents LPA from binding to numerous possible low affinity proteins, and suppresses LPA activation of endothelial cells, platelets, and circulating leukocytes (Figs. 1 and 2). At the lower concentrations of gelsolin observed in fluids of injured or inflamed tissues, which range from 1% to 10% of that in plasma,² its second function may be delivery of LPA to myocytes and some other types of cells with a greater effectiveness than that characteristic of serum albumin (Figs. 1 and 2). Confirming investigations by Stossel and co-workers² have characterized delivery of LPA to Swiss 3T3 cells by recombinant human gelsolin added to plasma from mice genetically totally deficient in gelsolin. LPA stimulation of the formation of actin stress fibers in 3T3 cells was enhanced maximally by gelsolin at approximately 1% normal plasma levels and was suppressed by normal plasma concentrations. The delivery of LPA to specific EdgRs by low concentrations of gelsolin may be driven by gradients of gelsolin concentration, which would be higher at the surface of cells producing gelsolin, as well as gradients of LPA affinity. That delivery of LPA to cellular receptors is the principal function of gelsolin in this context was shown by its lack of effect on immunospecific stimulation. RCMs express functional EdgRs for both LPA and S1P (Fig. 4; Table I). The nuclear signaling to SRE by a combination of phorbol ester and monoclonal anti-Edg-4 antibody at separately inactive concentrations was unaffected by gelsolin, which is consistent with the absence of any direct receptor or post-receptor effect of gelsolin (Table II).

Maximum stimulation of nuclear responses and enhancement of protein synthesis in RCM were attained when LPA was delivered by gelsolin at concentrations of 10% or lower than that in normal plasma (Figs. 1 and 2). Leakage of cytoplasmic gelsolin from ischemic or otherwise injured RCM and exudation of plasma gelsolin from permeabilized vasculature into inflamed myocardium at low concentrations thus initially would enhance locally the capacity of LPA to suppress cardiomyocyte apoptosis (40) and to evoke restorative events. Under different myocardial conditions, low levels of gelsolin may enhance LPA induction of greater contractility and/or promote LPA stimulation of myocardial hypertrophy. Of note, the fold increase in protein synthesis evoked by 100 nM LPA with 3 µg/ml gelsolin is greater than that induced by 1 µM norepinephrine or 1% fetal calf serum in other studies (29). However, at gelsolin concentrations above 20% those in normal plasma, later in the evolution of tissue responses plasma gelsolin may trap LPA, prevent effective delivery to EdgRs, and thereby diminish many of the biological effects of LPA. Thus gelsolin has the capacity to enhance and suppress the effects of LPA on myocytes and possibly other types of cells.

Molar excesses of PIP2 and substituent peptides of gelsolin constituting the PIP2-binding sites blocked LPA binding to gelsolin by 50% or more and inhibited LPA stimulation of both nuclear responses and protein synthesis (Fig. 6). This is consistent with a previous report of LPA regulation of the actin- binding, and -cleaving activity of gelsolin, which was similar to the effect of PIP2 and presumed to be attributable to LPA interactions with PIP2 sites (12). Gelsolin did not bind S1P nor deliver S1P to RCMs. Other plasma and non-receptor cellular proteins may bind S1P with effects similar to those of gelsolin for LPA but have not yet been identified. The mechanisms by which gelsolin delivers LPA to cells effectively remain to be elucidated but may include a gelsolin-selective docking site as well as bivalent and, thus, higher net affinity presentation. Also remaining for future studies are questions of any protective or carrier functions of gelsolin for LPA and any ability of gelsolin to facilitate transport of LPA into cells or across tissue boundaries.

	ACKNOWLEDGEMENT

We are grateful to Bethann Easterly for expert preparation of graphics.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants HL31809 (to E. J. G.) and HL54253 (to T. P. S.) and a Merit Review grant from the Department of Veterans Affairs Research Service (to J. S. K.). These data were presented in part at the 1999 annual meeting of the American Society for Biochemistry and Molecular Biology (41).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.

To whom correspondence should be addressed: University of California, UB8B, Box 0711, 533 Parnassus, San Francisco, CA 94143-0711. Tel.: 415-476-5339; Fax: 415-476-6915; E-mail: egoetzl@itsa.ucsf.edu.

² M. May, C. Lilly, K. Haley, M. Sunday, C. Sheils, P. Allen, T. P. Stossel, and J. M. Drazen, personal communication.

	ABBREVIATIONS

The abbreviations used are: LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; PIP2, L--phosphatidylinositol 4,5-bisphosphate; EdgR, endothelial differentiation gene-encoded G protein-coupled receptor; RCM, rat cardiac myocyte; DMEM⁺, Dulbecco's modified minimal Eagle's medium with penicillin and streptomycin; SRE, serum response element; faf-BSA, fatty acid-free bovine serum albumin; PMA, phorbol myristate acetate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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