Arachidonic acid (AA) liberated from membrane phospholipids is known to activate phospholipase C γ1 (PLCγ1) concurrently with AHNAK in nonneuronal cells. The recruitment of AHNAK from the nucleus is required for it to activate PLCγ1 at the plasma membrane. Here, we identify the C-type natriuretic peptide receptor (NPR-C), an atypical G protein-coupled receptor, as a protein binding partner for AHNAK1 in various cell types. Mass spectrometry and MASCOT analysis of excised bands from NPR-C immunoprecipitation studies revealed multiple signature peptides corresponding to AHNAK1. Glutathione S-transferase (GST) pulldown assays using GST- AHNAK1 fusion proteins corresponding to each of the distinct domains of AHNAK1 showed the C1 domain of AHNAK1 associates with NPR-C. The role of NPR-C in mediating AA-dependent AHNAK1 calcium signaling was explored in various cell types, including 3T3-L1 preadipocytes during the early stages of differentiation. Sucrose density gradient centrifugation studies showed AHNAK1 resides in the nucleus, cytoplasm, and at the plasma membrane, but small interfering RNA (siRNA)-mediated knockdown of NPR-C resulted in AHNAK1 accumulation in the nucleus. Overexpression of a portion of AHNAK1 resulted in augmentation of intracellular calcium mobilization, whereas siRNA-mediated knockdown of NPR-C or AHNAK1 protein resulted in attenuation of intracellular calcium mobilization in response to phorbol 12-myristate 13-acetate. We characterize the novel association between AHNAK1 and NPR-C and provide evidence that this association potentiates the AA-induced mobilization of intracellular calcium. We address the role of intracellular calcium in the various cell types that AHNAK1 and NPR-C were found to associate.
- smooth muscle cells
- rat gastric muscosa cell 1
- phospholipase C
calcium is a ubiquitous second messenger that plays an important role in the regulation of a wide range of cellular processes. Calcium release from intracellular stores occurs after the second messenger inositol 1,4,5-trisphosphate (IP3) is generated from the hydrolysis of phosphatidylinositol bisphosphate and binds to its receptor following activation of phospholipase C (PLC). At least four subfamilies of PLC have been identified and each is differentially regulated.
Activation of PLC-γ isozymes may occur through tyrosine-dependent or independent phosphorylation (35). Unsaturated fatty acids, such as arachidonic acid (AA), in concert with an extraordinary large phosphoprotein termed AHNAK, are known to activate PLC-γ1 (22, 35, 37). The AHNAK family of proteins AHNAK1 and AHNAK2 are structurally similar and consist of a short amino terminal domain, a large central domain with highly conserved repeated segments, and an isoform-specific carboxy terminal domain (16). Both AHNAKs appear to associate with calcium channel proteins of cardiomyocytes (21), although less is known about AHNAK2. The carboxy terminal domain of AHNAK1 contains nuclear export and localization signals (16, 44) and is responsible for its translocation and subcellular localization (30). AHNAK1 has been implicated in numerous cell-type-specific functions including T cell calcium signaling (24, 25), regulation of calcium channel gating properties in cardiomyocytes (1), modulation of DNA ligase IV-mediated double-stranded ligation (43), stabilization of skeletal muscle contractility (17), skeletal muscle regeneration (18), and plasma membrane differentiation and repair (4).
Activation of PLC-β isozymes occurs through stimulation of G protein-coupled receptors (GPCRs) and is mediated by the α-subunits of the Gq subfamily of heterotrimeric G proteins or by G protein βγ-subunits (35). The C-type natriuretic peptide receptor (NPR-C) is a single transmembrane protein that has recently been implicated in the activation of PLC-β3 (28). NPR-C functions as a homodimer and plays an important role in the clearance of natriuretic peptides (NPs) from circulation (23). Unlike the guanylyl cyclase-coupled natriuretic peptide receptors (NPRs)-A (NPR-A) and -B (NPR-B), NPR-C is widely expressed (5, 8, 12, 13, 45–47) and binds NPs with similar affinity (36). NPR-C consists of a short 37 amino acid cytoplasmic domain that has been reported to contain G protein-activating sequences (33, 49) to allow for inhibitory guanine nucleotide regulatory protein (Gi)-dependent signal transduction (28). NPR-C lacks the seven transmembrane motif of typical GPCRs and may therefore be considered an atypical GPCR. Since the role of NPR-C in signal transduction has not been fully characterized, we investigated whether NPR-C is associated with any scaffolding/adaptor proteins.
In this report, we present and characterize a novel association between AHNAK1 and NPR-C and elucidate the functional significance of this association in different cell types. We hypothesize NPR-C anchors AHNAK1 to the plasma membrane to facilitate its interaction with AA and proteins, including PLCγ1 and protein kinase C (PKC) to potentiate intracellular Ca2+ mobilization.
Reagents and antibodies.
Glass-bottom culture dishes were purchased from MatTek (Ashland, MA). All other cell culture plastic ware was purchased from Nalge Nunc (Rochester, NY). Precast gels (7.5% resolving with 4% stacking) were purchased from Bio-Rad (Hercules, CA). Protein G agarose was purchased from Upstate Biotechnologies (Lake Placid, NY). Mammalian protein extraction reagent (M-PER), Halt protease and phosphatase inhibitor, Super Signal West Pico chemiluminescent substrate, and BCA reagents were purchased from Pierce (Rockford, IL). Fluo-3 AM dye was purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted. Gαi antibody was kindly provided by Dr. Dave Manning (University of Pennsylvania, Philadelphia, PA); NPR-A and NPR-B polyclonal antibodies were kindly provided by Dr. David L. Garbers (University of Texas Southwestern, Dallas, TX); polyclonal AHNAK antibody was kindly provided by Dr. Hannelore Haase (Max Delbrück Center, Berlin, Germany); monoclonal AHNAK antibody was purchased from Abnova (Taipei City, Taiwan); β-actin antibody was purchased from Sigma; and PLCβ3, PLCγ1, PLCγ2, GAPDH, Lamin A/C, and peroxisome proliferator-activated receptor-γ (PPARγ) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-NPR-C antibody (JAH84) was from our laboratory. Hybond-C extra nitrocellulose membranes and Hyperfilm ECL were purchased from Amersham Biosciences (Piscataway, NJ).
Cell lines and cell culture.
3T3-L1 and HeLa cells were purchased from the American Type Culture Collection (Manassas, VA). HeLa cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM). Human aortic vascular smooth muscle cells (AoSMC) were purchased from Lonza (Walkersville, MD) and cultured in smooth muscle basal medium (Lonza). Rat gastric mucosa cells (RGM1) were acquired from Dr. Hirofumi Matsui (Riken, Japan) and cultured in DMEM/F12 medium. AoSMC, HeLa, and RGM1 cells were maintained at 37°C in a 5% CO2 humidified atmosphere. Cells were cultured in the presence of an antibiotic-antimycotic mixture and supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals; Norcross, Atlanta, GA) unless otherwise noted. All other cell culture media, calcium-free Hanks' balanced salt solution (HBSS), and antibiotic-antimycotic mixtures were purchased from GIBCO Invitrogen (Grand Island, NY). Only low passage cells were used for experiments.
3T3-L1 preadipocyte differentiation.
3T3-L1 preadipocytes were cultured in high-glucose DMEM supplemented with 10% newborn calf serum (NCS) (Sigma) and maintained at 37°C in a 10% CO2 humidified atmosphere. To induce differentiation, confluent 3T3-L1 preadipocytes were treated with 10 μg/ml insulin, 0.25 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine; cultured in high-glucose DMEM supplemented with 10% FBS; and maintained at 37°C in a 10% CO2 humidified atmosphere. Culture medium was replaced 48 h after differentiation with fresh culture medium containing insulin and 10% FBS.
Oil Red O staining.
3T3-L1 cells cultured on chamber slides were fixed with 10% formalin in PBS, washed with 1× PBS, and stained for 1 h at room temperature (RT) with 0.15% Oil Red O (60:40 mix of isopropanol and water). Slides were evaluated microscopically for accumulation of lipid droplets.
Recombinant fusion protein production.
cDNA fragments encoding various AHNAK1 domains were a kind gift from Dr. Takashi Hashimoto (Keio University School of Medicine, Tokyo, Japan). Domains were AHNAK1 N (residues 2-252), AHNAK1 M (residues 821-1330), AHNAK1 C1 (residues 4646-5145), and AHNAK1 C2 (residues 5146-5643). GST-AHNAK1 N, GST-AHNAK1 M, GST- AHNAK1 C1, and GST-AHNAK1 C2 constructs were kindly provided by Dr. Silvère M. van der Maarel (Leiden University Medical Center, Leiden, The Netherlands) (18). All constructs were confirmed by DNA sequencing. Plasmids were transformed into competent Escherichia coli BL21 (DE3) (Amersham Biosciences) for expression. Positive clones were grown in 2XYT kanamycin medium (16 g/l tryptone, 10 g/l yeast extract, and 5 g/l NaCl, pH 7.0) at 37°C. Isopropyl-d-thiogalactoside induction was performed at an optical density (OD600) of 0.4 by adding isopropyl-d-thiogalactoside to a final concentration of 0.01 M. GST-AHNAK N was obtained by traditional methods, and GST-AHNAK M, C1, C2 were solubilized from inclusion bodies. Bacterial cells were lysed by sonication, and the fusion protein was batch purified using glutathione Sepharose 4B. Purity of the fusion protein was analyzed by SDS-PAGE and Coomassie blue staining. Identity of the fusion protein was confirmed by Western blot analysis and by mass spectrometry analysis (Proteomics Dept. at the Moffitt Cancer Center, Tampa, FL).
SDS-PAGE, Western blotting, and densitometric analysis.
Total protein (25 μg) was subjected to SDS-PAGE, Western blotting, and densitometric analysis as previously described (5, 8, 13, 45–47). Blots were blocked in 5% nonfat dry milk in TBS at RT for 60 min. Anti-AHNAK1 and anti-NPR-B antibodies were used at a dilution of 1:500; anti-NPR-A antibody was used at a dilution of 1:4,000; and anti-NPR-C, PLCβ3, PLCγ1, PLCγ2, GAPDH, Lamin A/C were used at a dilution of 1:1,000 in 5% BSA in TBS at 4°C overnight. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Bio-Rad) was used at a dilution of 1:3,000 in 5% nonfat dry milk in TBS at RT for 60 min.
Total protein (200 μg) was incubated with a 1:250 dilution of anti-NPR-C or anti-AHNAK1 polyclonal antibody at 4°C for 2–4 h with end-over-end mixing. Complexes were incubated with a 1:10 dilution of prewashed 50% protein G agarose slurry (Upstate Biotechnology) at 4°C for 4–6 h with end-over-end mixing. Beads were washed 4× with ice-cold M-PER. Immune complexes were eluted in 1× SDS sample buffer and analyzed by SDS-PAGE and Coomassie staining or Western blotting.
GST pulldown assay.
Total protein (200 μg) was incubated with 25 μg of purified GST-AHNAK1 N, M, C1, or C2, or 25 μg of purified GST protein at 4°C for 2 h with end-over-end mixing. The resulting complexes were incubated with 40 μl of prewashed 75% glutathione Sepharose slurry (Amersham Biosciences) at 4°C for 4 h with end-over-end mixing. Beads were washed 4× with 1 ml of ice-cold GST lysis buffer [200 mM NaCl, 20 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), 0.5% Igepal CA-630, 25 μg/ml phenylethylsulfonyl fluoride, 2 μg/μl aprotinin, 1 μg/μl leupeptin, and 0.7 μg/ml pepstatin]. Bound proteins were eluted with 50 μl of 20 mM reduced glutathione in 50 mM Tris-Cl (pH 8.0) and then analyzed by SDS-PAGE and Western blotting.
Confocal fluorescence imaging and measurement of intracellular Ca2+.
Cells were cultured in complete growth media (containing calcium and NCS or FBS) in 35-mm glass-bottom culture dishes. At 70% confluency, cells were made quiescent by serum deprivation for 6 h. Cells were washed with calcium-free HBSS, loaded with the calcium-sensitive Fluo-3 AM dye at a final concentration of 10 μM in complete growth media, and incubated at 37°C for 20 min. Cells were washed with calcium-free HBSS and returned to complete growth media unless otherwise noted for 10 min to allow for deesterification of the dye. A confocal imaging system (Leica) equipped with an inverted microscope, an Argon 488 laser, and a ×63 1.4 numerical aperature objective lens was used to record calcium fluorescence at RT. Images were acquired at a rate of 1 frame every 2.5 s and recorded with LASAF software. A field of at least eight cells was chosen and focused in differential interference contrast mode. After baseline fluorescence was measured for 2 min, 100 μl of vehicle was added directly to the extracellular solution and fluorescence intensity was measured for an additional 2 min. Thereafter, 100 μl of the test agent was added and fluorescence intensity was measured for 2 min. Cells were then stimulated with 10 μM ionomycin to confirm equivalent loading.
Sucrose density gradient centrifugation.
Confluent 3T3-L1 preadipocytes were harvested from 100-mm tissue culture dishes for protein in TES buffer (20 mM Tris·HCl pH 7.4, 1 mM EDTA, and 255 mM sucrose) supplemented with protease inhibitors. The protein lysate was subjected to moderate sonication and then centrifuged at 17,000 g for 45 min at 4°C. The supernatant was collected and contained the cytosolic fraction. The pellet was resuspended in 0.5 ml TES buffer and layered on 0.3 ml of a 1.12 M sucrose cushion and then centrifuged at 250,000 g for 30 min at 4°C in a TL 55 swinging bucket rotor. The resulting supernatant layer was carefully removed and discarded. The intermediate layer was carefully collected and contained the membrane fraction. The bottom layer was then carefully removed and discarded. The pellet was resuspended in 100 μl of TES buffer and contained the nuclear fraction. Each fraction was quantified by the BCA assay and subjected to SDS-PAGE and Western blot analysis using specific subcellular marker antibodies.
Transfection of siRNA and constructs.
3T3-L1 preadipocytes were transfected with siGenome SMARTpool reagents (Dharmacon; Lafayette, CO) specific for mouse NPR-C, mouse AHNAK1, or nontargeting siRNA using DharmaFECT3 transfection reagent (Dharmacon) according to the manufacture's instructions. Protein knockdown was determined by Western blot analysis and densitometric analysis 72 h after transfection. 3T3-L1 preadipocytes were transfected with pBIND- AHNAK1, composed of the C1 domain and several central repeating units of AHNAK1, using FuGENE 6 (Roche; Indianapolis, IN), according to the manufacturer's instructions. Transfection efficiency was assessed by luciferase assays 72 h after transfection.
Results are expressed as means ± SE of three independent experiments. Statistical significance was set at P < 0.05. *P > 0.05, **P < 0.005, ***P < 0.0005. Student's t-test was used to compare two groups and one-way ANOVA was used to compare multiple groups.
AHNAK1 associates with NPR-C.
As increasing evidence suggests NPR-C plays a role in signaling, we investigated putative protein binding partners for NPR-C that could help explain its role in mediating signal transduction. We immunoprecipitated NPR-C from AoSMC lysate, separated the eluted proteins by SDS-PAGE, and analyzed the immunoprecipitants from Coomassie blue-stained gels. NPR-C (Fig. 1A) and AHNAK1 (data not shown)-specific antibody resulted in the pulldown of a high-molecular-weight double band that was absent with protein G agarose beads alone (Fig. 1A) or with nonspecific antibody (data not shown). Mascot results of the excised double band revealed multiple signature peptides corresponding to human AHNAK isoform 1 (Fig. 1B). Western blot analysis confirmed AHNAK1 as the immunoprecipitated protein from AoSMC lysates (Fig. 1C). Similar results were seen when using RGM1 (Fig. 1D) and 3T3-L1 preadipocyte lysates (Fig. 1E). Mascot results of a high-molecular-weight double band from Coomassie blue-stained gels after immunoprecipitating with NPR-C antibody and using RGM1 lysate revealed multiple signature peptides corresponding to rat AHNAK isoform 1 (data not shown). Thus AHNAK1, and not AHNAK2, appears to be a protein binding partner for NPR-C in various cell types.
C1 domain of AHNAK1 associates with the cytoplasmic tail of NPR-C.
To corroborate the association between AHNAK1 and NPR-C and to determine the specific domain of AHNAK1 that associates with NPR-C, we synthesized and purified GST fusion proteins representative of each of the distinct domains of AHNAK1 (Fig. 2A), AHNAK1 N (amino terminal residues 2-252), AHNAK1 M (central repeat unit residues 821-1330), and two carboxy terminal domains (AHNAK1 C1 residues 4646-5145, and AHNAK1 C2 residues 5146-5643). Verification of molecular weight and purity was determined by SDS-PAGE followed by Coomassie blue staining (Fig. 2B) and by direct Western blot analysis for GST (Fig. 2C). We then performed an in vitro GST pull-down assay using cell lysate from AoSMCs and the purified GST-AHNAK1 fusion proteins. Only GST-AHNAK1 C1 was able to pulldown NPR-C (Fig. 2D). In similar experiments, GST-AHNAK1 C1 was not able to pulldown NPR-A or NPR-B (data not shown), as NPR-A and NPR-B specific antibodies also failed to pulldown AHNAK1 (data not shown). Accordingly, it is the unique cytoplasmic domain of NPR-C that associates with the C1 domain of AHNAK1.
Endogenous expression of AHNAK1, NPRs, Gαi, and PLC isoforms.
Both NPR-C and AHNAK1 proteins have been implicated in the activation of PLC, albeit different isoforms. Figure 3 shows endogenous protein expression of AHNAK1, NPRs, Gαi, and PLC isoforms in the various cell lines that we used to demonstrate the AHNAK1 and NPR-C association. Relative protein expression levels are shown for AHNAK1 and NPRs in Fig. 3A and for Gαi in Fig. 3B. HeLa, AoSMC, RGM1, and 3T3-L1 preadipocytes were found to express varying levels of PLCγ1 (Fig. 3C) and PLCβ3 (Fig. 3E) but no appreciable levels of PLCγ2 (Fig. 3D). Thus activation of the PLCγ2 pathway cannot be implicated when considering the functional significance of the association between AHNAK1 and NPR-C in AoSMC, RGM1, or 3T3-L1 preadipocytes.
Subcellular localization of AHNAK1 is influenced by NPR-C knockdown.
The translocation of AHNAK1 from the nucleus to the cytoplasmic region of the plasma membrane is required for activation of PLCγ1 by AHNAK1 in conjunction with AA (37). We evaluated the effect of NPR-C knockdown by siRNA on the translocation and subcellular localization of AHNAK1 protein in 3T3-L1 preadipocytes. In three similar independent experiments, Western blot and densitometric analysis showed there was at least a 70% knockdown of NPR-C protein by siRNA (Fig. 4, A and B), which resulted in approximately a 40% decrease in AHNAK1 protein associated with the membrane fraction (Fig. 4, C and D) and approximately a 40% increase in AHNAK1 protein associated with the nuclear fraction (Fig. 4, C and D). Taken together, NPR-C appears to recruit and anchor AHNAK at the plasma membrane of 3T3-L1 preadipocytes.
AHNAK1 and NPR-C are differentially expressed during 3T3-L1 differentiation.
In addition to being tolerant to transient transfection, the 3T3-L1 cell line is a commonly used mouse model for the study of adipogenesis. Therefore, we investigated the regulation of NPR-C and AHNAK1 proteins during the differentiation of 3T3-L1 preadipocytes to mature adipocytes. The progression of the differentiation was monitored every other day by phase-contrast microscopy and Oil Red O staining for the accumulation of lipid droplets (Fig. 5, A and B). At each corresponding day, protein expression of the NPRs, AHNAK1, and PPARγ were assessed by Western blot analyses. There were no significant changes in NPR-A or NPR-B protein expression during the differentiation for days 0–6 (data not shown). However, AHNAK1 protein expression significantly decreased (Fig. 5, D and E), and NPR-C protein expression significantly increased from days 0–6 (Fig. 5, F and G).
Next, we confirmed the differential regulation of AHNAK1 and NPRs proteins in mouse adipose tissue. We probed adipose tissues from three different mouse donors for AHNAK1 and NPRs. Western blot analysis indicated low protein expression levels for NPR-A and NPR-B, abundant protein expression for NPR-C, and no appreciable protein expression for AHNAK1 (Fig. 5H). Similar trends were consistently observed in mature mouse adipocytes coresponding to days 8–12 of 3T3-L1 differentiation (data not shown). Therefore, expression of AHNAK1 and NPR-C appears to be inversely proportional in the late stages of differentiation, whereas both proteins are expressed in the early stages of differentiation of 3T3-L1 preadipocytes.
Exogenous AA increases intracellular Ca2+.
We examined whether exogenous AA could induce intracellular Ca2+ mobilization in a dose-dependent manner in 3T3-L1 preadipocytes and in AoSMCs in situ. Although there was not a significant response with the addition of 1 μM of exogenous AA (data not shown), a rapid and transient increase in intracellular Ca2+ within 30 s was observed with the addition of 25 μM (Fig. 6A) and 100 μM (Fig. 6B) of exogenous AA in 3T3-L1 preadipocytes. AoSMC also responded within 30 s to the addition of 25 and 100 μM of exogenous AA (data not shown). The addition of vehicle alone failed to elicit a response in either 3T3-L1 preadipocytes or in AoSMCs. Thus AA is able to induce a rapid and transient increase in intracellular calcium in cultured 3T3-L1 preadipocytes and AoSMCs.
AA-mediated increases in intracellular Ca2+ is from release from intracellular Ca2+ stores.
The source of the increase in intracellular Ca2+ that was observed from the addition of exogenous AA was then investigated. To identify whether the increase of intracellular Ca2+ was attributed to an influx of calcium from the extracellular medium or release of calcium from intracellular Ca2+ stores, 3T3-L1 preadipocytes and AoSMC (data not shown) were treated and monitored in the presence or absence of Ca2+ in the extracellular medium. For both 3T3-L1 preadipocytes (Fig. 6) and AoSMC (data not shown), the addition of exogenous AA resulted in a rapid and transient increase in intracellular calcium in cells cultured in the presence (Fig. 6A) or absence (Fig. 6, B and C) of calcium in the extracellular medium. Consequently, the rapid and transient increase in intracellular Ca2+ observed from the addition of exogenous AA in 3T3-L1 preadipocytes or AoSMCs is from release of calcium from intracellular stores and not an influx of extracellular calcium.
Phorbol 12-myristate 13-acetate increases intracellular Ca2+ in 3T3-L1 preadipocytes overexpressing an AHNAK1 construct.
It has been reported that NIH 3T3 cells release AA in response to phorbol 12-myristate 13-acetate (PMA) treatment (22). The addition of PMA induced AHNAK1 interaction with PLC-γ1 and mobilization of intracellular Ca2+ in those cells. Therefore, we investigated whether PMA had a similar effect in 3T3-L1 preadipocytes in a dose-dependent manner. In 3T3-L1 preadipocytes, PMA treatment resulted in a rapid and transient increase in intracellular Ca2+ in situ (Fig. 7).
To confirm AHNAK1 was involved in the mobilization of intracellular Ca2+, we created an AHNAK1 fusion protein that consisted of the carboxy terminal domain and several central repeating units of AHNAK1, along with a luciferase reporter gene to measure transfection efficiency. Seventy-two hours after transient transfection of this construct into 3T3-L1 preadipocytes, we measured intracellular Ca2+ in response to the addition of PMA. When compared with untransfected 3T3-L1 preadipocytes (Fig. 7A), the increase in intracellular Ca2+ in response to PMA treatment was significantly augmented in 3T3-L1 preadipocytes overexpressing the AHNAK1 fusion protein (Fig. 7, B and G).
Next, we verified the mechanism of PMA in the activation of cPLA2 and subsequent liberation of endogenous AA from membrane phospholipids in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were preincubated with the cPLA2 inhibitor AACOCF3, and PMA was added to induce an increase in intracellular Ca2+. When compared with untreated 3T3-L1 preadipocytes (Fig. 7A), the increase in intracellular Ca2+ in response to PMA treatment was significantly attenuated in 3T3-L1 preadipocytes pretreated with the cPLA2 inhibitor (Fig. 7, C and G).
Thus, in 3T3-L1 preadipocytes, AHNAK1 appears to serve as a receptor for AA, and together they are involved in intracellular Ca2+ mobilization. Also, in 3T3-L1 preadipocytes, PMA is capable of activating cPLA2, resulting in the release of AA from membrane phospholipids and subsequent intracellular Ca2+ mobilization in the presence of AHNAK1.
Knockdown of NPR-C or AHNAK1 attenuates the increase of PMA-induced intracellular Ca2+ in 3T3-L1 preadipocytes.
Next, we investigated a putative role for NPR-C in the AHNAK1-mediated and AA-dependent mobilization of intracellular Ca2+ in 3T3-L1 preadipocytes. We decreased the NPR-C protein level by siRNA knockdown in 3T3-L1 preadipocytes, and after 72 h we measured intracellular Ca2+ in response to the addition of PMA. When compared with untransfected 3T3-L1 preadipocytes (Fig. 7A), the increase in intracellular Ca2+ in response to the addition of PMA was significantly attenuated in 3T3-L1 preadipocytes, in which the NPR-C protein level was decreased by siRNA knockdown (Fig. 7, D and G). Similarly, the increase in intracellular Ca2+ in response to the addition of PMA was significantly attenuated in 3T3-L1 preadipocytes, in which the AHNAK1 protein level was decreased by siRNA knockdown (Fig. 7, E and G).
To show NPR-C by itself was not responsible for the increases in intracellular Ca2+ observed in 3T3-L1 preadipocytes, we activated NPR-C with the specific agonist des[Gln18, Ser19, Gly20, Leu21, Gly22]-ANP(4-23)-NH2 (cANF) and then monitored changes in intracellular Ca2+. When compared with the addition of vehicle, the addition of cANF did not result in an increase in intracellular Ca2+ (Fig. 7F). Therefore, in 3T3-L1 preadipocytes, NPR-C potentiates intracellular Ca2+ mobilization in the presence of AHNAK1 and AA independent of receptor occupancy by ligand.
In this report, we have provided evidence for a novel cell type-dependent association between AHNAK1 and NPR-C. We have shown a role for NPR-C in the recruitment of AHNAK1 to the plasma membrane, where AHNAK1 is known to form a complex with various proteins and mediate calcium signaling. In addition, we have identified the differential regulation of AHNAK1 and NPR-C proteins during mouse adipogenesis and determined the functional significance of their association in 3T3-L1 preadipocytes during the early stages of differentiation. The increase in intracellular Ca2+ in response to PMA treatment in mock transfected 3T3-L1 preadipocytes was augmented in cells transfected with an AHNAK1 construct and attenuated in cells transfected with NPR-C or AHNAK1-specific siRNAs. The source of increase in intracellular Ca2+ in response to PMA was due to release of Ca2+ from intracellular stores.
We screened multiple cell types for endogenous AHNAK1 and NPR-C protein expression to investigate the molecular determinants and physiological significance of the association between AHNAK1 and NPR-C. AHNAK1 protein was identified as a protein marker of cells with barrier properties (9). Epithelial cells of the gastric mucosa form a barrier against luminal contents. An epithelial cell line, originating from normal rat gastric mucosa (RGM1) (20), was used as a model to investigate the molecular association between AHNAK1 and NPR-C (Fig. 1D). Furthermore, RGM1 cells express mRNA for NPR-C (12) and abundantly express endogenous NPR-C protein but do not express any appreciable levels of NPR-A or NPR-B proteins (Fig. 3A). Since there are numerous reports of NPR-C signaling in vascular smooth muscle cells (SMC) (2) and AHNAK1 expression has already been described in SMC (10), we corroborated the association between AHNAK1 and NPR-C using a AoSMC line. However, because we experienced difficulty in transfecting the RGM1 and AoSMC cell lines by traditional methods, we also evaluated a mouse 3T3-L1 cell line as a model to investigate the physiological significance of the molecular association between AHNAK1 and NPR-C in situ. In addition to the 3T3-L1 cell line being transfectable, it is a well-characterized model for the study of adipocyte differentiation (14, 15). The expression of AHNAK1 protein has been reported in NIH 3T3 cells (42). Consistent with reports by others (11, 19), we found abundant levels of NPR-C protein expression in rodent adipocytes. Another report indicated NPs induced lipolysis specifically in primates (38). Here, we show NPR-C protein is the only NPR subtype to significantly increase with mouse preadipocyte differentiation (Fig. 5, F and G). AHNAK1 protein expression appeared to be downregulated or susceptible to proteolysis with mouse preadipocyte differentiation, as indicted by the absence of upper molecular weight bands corresponding to AHNAK1 in Western blots for days 0 to 6, and the presence of lower molecular weight bands (Fig. 5, D and E). Consistent with this finding, in separate experiments (data not shown), we observed lower molecular weight bands corresponding to AHNAK1 protein in the absence of protease inhibitors, further suggesting AHNAK1 protein is readily susceptible to proteolysis with differentiation of 3T3-L1 preadipocytes. The inversely regulated protein expression levels between AHNAK1 and NPR-C during differentiation of 3T3-L1 cells explains our observation of a direct association between AHNAK1 and NPR-C in 3T3-L1 preadipocytes during the early stages of differentiation but not in mature adipocytes during the later stages of differentiation. These findings also suggest the association between AHNAK1 and NPR-C is transient and cell-type dependent.
Although the expression of AHNAK1 has been reported to increase upon retinoic acid-induced differentiation of neuroblastoma cells, the function of AHNAK1 was described to be specialized rather than universal (41). Our observations of AHNAK1 protein being expressed in 3T3-L1 preadipocytes during the early stages of differentiation and suppressed in the later stages of differentiation suggest AHNAK1 may serve an important role in mouse adipogenesis.
The role of AHNAK1 in the activation of PLCγ1 and intracellular Ca2+ homeostasis has been well documented (22, 37). AHNAK1 has been shown to activate PLCγ1 in the presence of AA (22, 37) and was shown to play a critical role in the regulation and function of cardiac Ca(v)1.2 calcium channels (16). Interestingly, NPR-C has also been implicated in the activation of PLC, albeit a different isoform than PLCγ1. NPR-C has been shown to activate PLCβ3 through Gαi (33) via β-subunits (27). We investigated the functional significance of the association between AHNAK1 and NPR-C downstream of PLC activation. We show evidence for the role of AHNAK1 and NPR-C in the mobilization of intracellular Ca2+ in response to exogenous AA or PMA-induced release of intracellular AA in 3T3-L1 preadipocytes. Cytosolic phospholipase A2 (cPLA2) catalyzes the release of AA from membrane phospholipids (6), and its activation and phosphorylation has been suggested to be regulated by PKC (29, 48). The PKC activator PMA was shown to release AA from cPLA2 activation in NIH 3T3 cells (22). PMA was also shown to augment the production of inositol phosphates and intracellular Ca2+ mobilization in NIH 3T3 cells overexpressing central repeating units of AHNAK1 (22). To determine whether AA could mobilize intracellular Ca2+ in 3T3-L1 preadipocytes, we treated quiescent Fluo-3-loaded cells with exogenous AA or PMA at various concentrations. Treatments with either exogenous AA or PMA resulted in a rapid and transient increase in intracellular Ca2+ in 3T3-L1 preadipocytes. We observed attenuation in the intracellular Ca2+ response to PMA when we pretreated 3T3-L1 preadipocytes with the AA analog and inhibitor of cPLA2, AACOCF3 (Fig. 7C), thus confirming the PMA-induced increase in intracellular Ca2+ was due to the release of AA from membrane phospholipids after cPLA2 activation.
The association between AHNAK1 and NPR-C at the plasma membrane may be dependent on several different events including the rate of internalized receptors recycling back to the cell surface, the turnover and abundance of receptors at the cell surface, and the stimulation of these receptors at the cell surface. The clearance function of NPR-C is well characterized. Upon ligand binding, the NPR-C-ligand complex undergoes endocytosis via clathrin-coated pits, which is thought to be dependent on the integrity of the cytoplasmic domain of NPR-C (7). The complex then dissociates intracellularly in endosomes, followed by hydrolysis of ligand in lysomes and rapid recycling of receptor back to the cell surface (32). Initially, we thought the association between AHNAK1 and NPR-C at the membrane would be attenuated by inducing NPR-C ligand binding, since a population of surface receptors would be internalized. However, we did not observe a significant change in the association between AHNAK1 and NPR-C after treating RGM1 cells with the NPR-C agonist cANF for various allotted time intervals (data not shown). This finding may be attributed to the rapid rate of NPR-C recycling back to the membrane and also suggest the association between AHNAK1 and NPR-C is independent of NPR-C activation by ligand binding. Alternative means of activation for cell-surface receptors have been documented. For example, deoxycholic acid can cause ligand-independent activation of epidermal growth factor receptor in primary hepatocytes (34). We believe the cytoplasmic domain of NPR-C may contain peptide sequences sufficient for AHNAK1 association. Zhou and Murthy (49) showed four synthetic peptides derived from the intracellular domain of NPR-C are capable of activating G proteins, and one of these peptides is sufficient for the selective activation of Gi1 and Gi2.
AHNAK1 is shown as a versatile protein that is able to translocate from the nucleus to the plasma membrane and associate with various proteins (Fig. 8). AHNAK1 is an extraordinary large protein but consists of four structural domains (16). The highly conserved central repeating units of AHNAK1 are thought to bind directly to PLCγ1 (37) and PKC (22). Here we show the C1 domain of AHNAK1 associates with NPR-C, although the binding motif containing the specific amino acid residues involved in the association between the two proteins have not yet been identified. Since AHNAK1 is associated with the cytoplasmic face of the plasma membrane upon translocation from the nucleus, we believe it is the cytoplasmic domain, and not the extracellular or transmembrane domain of NPR-C, that associates with AHNAK1. We demonstrated NPR specificity for the association with AHNAK1 protein as NPR-C (Fig. 1) and not NPR-A or NPR-B (data not shown) co-immunoprecipitated with AHNAK1. Similarly, GST-AHNAK1 C1 was able to pulldown NPR-C (Fig. 2) but not NPR-A or NPR-B (data not shown). We hypothesize the functional role of NPR-C in its association with AHNAK1 is to tether AHNAK1 to the plasma membrane to facilitate its interaction with PLCγ1 and PKC. The tethering of AHNAK1 by NPR-C to the plasma membrane also allows AHNAK1 to be in close proximity to AA for activation of PLCγ1. To our knowledge this is the first report of a cell surface receptor associating with AHNAK1. However, it is likely other transmembrane proteins may also recruit or interact with AHNAK1 at the cell surface, since increasing evidence suggest AHNAK1 functions at the plasma membrane.
The concentration of intracellular Ca2+ is maintained at low levels (50–100 nM) in most cells (3), and it is conceivable that cells are sensitive to even slight changes in intracellular Ca2+. Moreover, the role of intracellular Ca2+ mobilization is cell-type dependent. For example, intracellular Ca2+ mobilization is crucial for vascular SMC contractility, and abnormal vascular SMC contractility may contribute to abnormal vascular tone and disorders of blood pressure regulation, including hypertension (26). In experiments with AoSMC, we also observed an increase in intracellular Ca2+ in response to exogenous AA (data not shown), which may also be mediated by the association between AHNAK1 and NPR-C. In this investigation, we propose a novel pathway to target the regulation of intracellular Ca2+ in 3T3-L1 preadipocytes. There is increasing evidence for the role of intracellular Ca2+ in the regulation of adipogenesis. Increasing levels of intracellular Ca2+ were reported to have an inhibitory effect in the early stages of differentiation of 3T3-L1 preadipocytes into adipocytes (31). Ntambi and Takova (31) suggested that calcium mobilization repressed the synthesis of an intermediate involved in DNA replication to prevent the expression of transcription factors required for differentiation. Increasing levels of intracellular Ca2+ has been implicated in the inhibition of differentiation in the early stages and promotion of differentiation in late stages of human adipocyte differentiation (40). Our results suggest NPR-C may potentiate AA-dependent AHNAK1 calcium signaling in the early stages of differentiation. However, additional studies will be necessary to determine whether a constitutively active calcium signaling pathway can inhibit adipogenesis in the early states of differentiation.
In summary, we provide evidence for the molecular association between AHNAK1 and NPR-C. We suggest the cytoplasmic tail of NPR-C tethers AHNAK1 at the plasma membrane to facilitate its association with AA and various proteins, which have already been reported. Our findings suggest an alternative route of PLC activation involving NPR-C and may have further implications for the NP system in calcium homeostasis. This study suggests the association between AHNAK1 and NPR-C can potentiate an AA-dependent increase in intracellular Ca2+ from release of intracellular stores in 3T3-L1 preadipocytes. Further investigations will be necessary to characterize the phenotype of these cells in response to an increase in intracellular Ca2+ induced by AA. Additional studies will also be necessary to better understand the role of the association between AHNAK1 and NPR-C in pathogenesis.
This work was supported by an American Heart Association Predoctoral fellowship (to A. A. Alli) and a Veterans Affairs Merit grant (W. R. Gower).
We thank Dr. Javier Cuevas for critically reviewing this manuscript. We thank Joseph O. Johnson for assistance with the confocal microscopy studies (Analytic Microscopy Core at the Moffitt Cancer Center, Tampa, FL).
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