Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide
© Chrisman et al; licensee BioMed Central Ltd. 2003
Received: 16 September 2003
Accepted: 19 November 2003
Published: 19 November 2003
Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s) is of significant medical importance.
Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2), a known component of high density lipoprotein (HDL). Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P). We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor) for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5–10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK) or Akt/protein kinase B (Akt/PKB).
Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this mitogenic signaling molecule likely functions as a strong adversary of the CNP/GC-B signaling pathway in the regulation of cell proliferation and other growth factor-induced phenotypes. The mechanism by which S1P desensitizes GC-B appears different than the known S1P signaling pathways.
Atrial natriuretic peptide (ANP1), C-type natriuretic peptide (CNP), and cell-permeant guanosine-3',5'-monophosphate (cGMP) analogs antagonize growth factor-stimulated proliferation and chemotaxis in cultured fibroblasts and vascular smooth muscle cells, and CNP also slows coronary artery restenosis in vivo [1–3]. Conversely, serum and certain peptide and phospholipid growth factors suppress CNP-elevations of cGMP in primary dermal fibroblasts, BALB-c/3T3 fibroblasts, or NIH/3T3 fibroblasts that over-express the receptor for CNP, guanylyl cyclase B (GC-B) [1, 4]. Therefore, it has been suggested that CNP/GC-B and various mitogens oppose each other in the regulation of tissue remodeling after injury [1, 3, 4]. Adult cardiac fibroblasts, for example, synthesize CNP and express GC-B and CNP or cell permeant analogs of cGMP antagonize increases in DNA and collagen synthesis promoted by serum [5, 6]. In previous studies, a number of growth factors and other agents have been shown to cause desensitization of guanylyl cyclase A (GC-A) or GC-B, possibly through the induction of receptor dephosphorylation [1, 7, 8]. As we compared the relative potencies of various serum growth factors (platelet-derived growth factor, PDGF; lysophosphatidic acid, LPA; basic fibroblast growth factor, bFGF) to desensitize GC-B, we realized that serum acted at dilutions where the above factors could not represent the most active desensitization factor. We then identified a serum factor (sphingosine-1-phosphate; S1P) that causes desensitization of GC-B at concentrations well within the range of those found in serum.
Identification of a Protein Component in Purified Inhibitory Fractions
Desensitization of GC-B by HDL
Extraction of lipid components into Butanol
Identification of Serum Factor
Desensitization in other Cells
Uniqueness of the Desensitization Pathway
A unifying model for the actions of various mitogens and S1P to desensitize GC-B appears to require the existence of receptors that are not coupled to Gi. These could be cell surface or intracellular receptors. In the model of Spiegel and colleagues [17, 21] PDGF or serum activate sphingosine kinase, which then catalyzes increased formation of S1P. S1P may then act on intracellular receptors and bypass the G-coupled receptors on the plasma membrane. Such a pathway would provide a unifying model to explain the common action of various mitogens to desensitize the CNP receptor independent of ERK or Akt activation. On the other hand, possibly the various mitogens, including S1P, act through a unique pathway not involving Gi or the activation of pathways such as those of ERK or Akt.
The contents of cGMP in cultured cell lines were determined as previously described. Human aortic smooth muscle cells were from Clonetics and cultured as instructed by the supplier. NIH/3T3 cells over-expressing rat GC-B (GCB/3T3) were as previously described ; continuous passaging of this cell line can result in decreased expression of GCB (Fig. 1vs Fig 7). Human HDL was from Biotechnology Industries and was diluted in phosphate-buffered saline (PBS) just prior to experimentation. Peptide sequencing was by the Mass Spectroscopy Facility at UT Southwestern Medical Center in Dallas. Pertussus toxin was from List Biochemicals and fetal bovine serum (FBS) was from Atlanta Biologicals.
Desensitization of GC-B to the ligand CNP was determined in duplicate or quadruplicate as described previously . Confluent cells in 24-well tissue culture plates were placed in serum-deficient DMEM for 24 to -48 h and were then transferred to fresh serum-deficient medium 2 h prior to experimental treatments. Stock solutions of CNP (Peninsula Biochemicals), LPA and S1P (Sigma Aldrich) were diluted in fatty acid-free bovine serum albumin (Sigma Aldrich) at 100-times the concentration in the cell reactions. Samples were added to the cells for 30 min prior to the addition of 0.25 mM 3-isobutyl-1-methylxanthine for 10 min; this was followed by the addition of CNP to a 20 nM final concentration for 5 min. Addition of perchloric acid to a final concentration of 0.5 N terminated the cell reactions. cGMP content of each well was determined in duplicate by radioimmunoassay as described . Bovine HDL (d = 1.063–1.21) was isolated from heat-inactivated fetal bovine serum (FBS) by centrifugation into KBr as described for isolation of human plasma HDL . The resulting preparations were at least 90% pure as judged by protein staining (GelCode Blue Stain Reagent (Pierce)) following sodium dodecylsulfate(SDS)-polyacrylamide gel electrophoresis.
Butanol extraction of HDL or partially purified serum factor
One mg human HDL in 0.8 ml PBS/20 mM acetic acid was mixed with 0.4 ml n-butanol/20 mM acetic acid and the phases separated by centrifugation as described by English et al. . Twenty-μl aliquots of the upper ("butanol') and lower ("water") phases were dried and dissolved in 50 μl PBS and 5 μl used for cell incubation. For the partially purified serum factor preparation, active fractions from the initial C18 column were dried and the residue dissolved in 4 ml PBS/20 mM acetic acid and 2 ml n-butanol/20 mM acetic acid added. Further processing was as described above except that the 50-μl aliquots were dried and dissolved in 500 μl PBS.
The phosphorylated forms of extracellular signal-regulated kinases 1 and 2 (pERK1/2) were determined by Western blot using rabbit antisera to pERKs (Promega) diluted 1:20,000 as previously described  with the modification that the horseradish peroxidase-linked secondary antibody (goat anti-rabbit IGG) was diluted 1:500,000 and chemiluminescence detected by Pierce SuperSignal West Femto reagent. Phosphorylated Akt/PKB (pAkt) was detected using rabbit antisera to phosphoserine 473 (Cell Signaling Technology) diluted 1:1000 and the secondary antibody diluted 1:250,000 and detected as above.
Purification of Desensitization Activity
Isolation of a desensitization component of heat-inactivated FBS was accomplished using a combination of anion exchange chromatography (DEAE) and C18-reverse-phase chromatography. Generally, three hundred ml serum was diluted with 4 volumes 25 mM Tris, pH 7.8–50 mM NaCl and mixed with 200 ml DEAE-Sepharose, equilibrated in the same buffer overnight at 8°C. The mixture was filtered, washed with 1000 ml 25 mM Tris, pH 7.8–100 mM NaCl and the washed DEAE Sepharose placed in a 5 cm diameter column. Protein was eluted from the DEAE-Sepharose with 500 ml 25 mM Tris, pH 7.8–200 mM NaCl and 10-ml fractions were collected. Fractions with an A280 greater than 1.0 were pooled (about 100 ml) and acetic acid and triflouroacetic acid (TFA) were added to final concentrations of 10% and 0.1%, respectively. The acidified fractions were applied to 3 C18 cartridges (Sep-Pak Plus C18, Waters) equilibrated in 0.1% TFA and each washed with 20 ml 40% acetonitrile-0.1% TFA prior to elution of retained material with 10-ml 60% acetonitrile-0.1% TFA per cartridge. The pooled eluates were diluted approximately 3-fold with 0.1% TFA and applied to a C18 column (Vydac 218TP510) equilibrated in 35% acetonitrile-0.1% TFA at a flow rate of 2-ml/min. The column was washed for 10 min with 35%acetonitrile-0.1% followed by 49% acetonitrile-0.1% TFA for 10 min and then a linear 49%–53% acetonitrile gradient over 40 min. Inhibitory activity in the 2-ml fractions was monitored using inhibition of CNP-elevated cGMP in the GC-B/3T3 cell line. The fractions eluting at approximately 51% acetonitrile contained significant desensitization activity. The active fractions were pooled, diluted 3-fold in 0.1% TFA and applied to an analytic C18 column (Vydac 218TP54) equilibrated in 5% acetonitrile-0.1% TFA followed with 48.8% % acetonitrile-0.1% TFA for 10 min and then with a linear 48.8%–50.3% acetonitrile-0.1% TFA gradient for 65 min. Active fractions eluting at approximately 50 % acetonitrile were diluted with 2 volumes 0.1% TFA and reapplied to the analytic C18 column equilibrated in 5% acetonitrile-0.1% TFA, washed for 5 min with 5% acetonitrile-0.1% TFA and then for 5 min with 48% acetonitrile-0.1% TFA. Desensitization activity was eluted with a linear 48–49% acetonitrile-0.1% TFA gradient obtained over 60 min at 1 ml/min. Active fractions contained a single symmetrical peak of absorbance at 280 nm that co-eluted with inhibitory activity (Fig 1).
List of Abbreviations
atrial natriuretic peptide
C-type natriuretic peptide
guanylyl cyclase B
guanylyl cyclase A
fetal bovine serum
extracellular regulated kinase
Akt/protein kinase B
guanylyl cyclase B/NIH 3T3 fibroblasts
high density lipoprotein
This work was supported in part by an award to DLG from the Sandler Program for Asthma Research, the Howard Hughes Medical Institute and the Cecil H. and Ida Green Center for Reproductive Biology Sciences.
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