Abstract
Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating APN is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of APN through the Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. APN expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by the detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on APN promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV-dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also canceled the effect of HDL on APN gene expression. These results suggest that HDL has an important role to improve the function of adipocytes by activating hSR-BI/CLA-1, and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.
Introduction
One of the main roles of adipocytes is to store energy as triglycerides during feeding and to provide free fatty acids to other tissues during fasting. It also plays an important role as an endocrine organ by secreting adipokines that contribute to the regulation of energy metabolism in the body. Adiponectin (APN), an adipokine, is an adipocyte-derived vasoactive peptide with anti-inflammatory and insulin-sensitizing peptides. A number of clinical trials have shown that subjects with high levels of circulating APN tend to be protected against type 2 diabetes and myocardial infarction (Kadowaki et al. 2006). It has been reported that higher concentrations of APN protect against the later development of type 2 diabetes (incidence rate ratio 0.63; 95% CI, 0.43–0.92, P = 0.02) (Dekker et al. 2008). Furthermore, participants in the highest are compared with the lowest quintile of APN levels and have been reported to have a significantly decreased risk of myocardial infarction (relative risk, 0.39; 95% CI, 0.23–0.64; P < 0.001) (Pischon et al. 2004).
APN has been correlated with various parameters of lipoprotein metabolism. Specifically, APN is negatively correlated with serum triglycerides and positively correlated with HDL-C (Christou & Kiortsis 2013). HDL exerts several effects on metabolic homeostasis and thereby beneficially impacts glucose homeostasis, inflammation, thrombosis, and atherosclerosis. HDL plays an important role in removing excess cholesterol from peripheral tissues and cells. This process of peripheral cholesterol efflux to HDL and delivery to the liver is termed reverse cholesterol transport and is important for the anti-atherogenic properties of HDL. Several clinical studies reported that circulating APN levels are positively correlated with HDL-C serum levels (Christou & Kiortsis 2013). HDL-C itself has been shown to be positively correlated with high-molecular weight (HMW) APN, which is considered the most biologically active fraction of APN (Kangas-Kontio et al. 2010). HMW APN has a high correlation coefficient, independent of various factors of adiposity and insulin sensitivity (Im et al. 2006).
The mouse scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL-C ester in transfected Chinese hamster ovary cells. This finding provides an important link between a specific cell surface receptor and a pathway involved in the uptake of HDL-C (Acton et al. 1996). Our previous studies show that CD36, and LIMPII analogous-1 (hSR-BI/CLA-1), like mouse SR-BI, functions as a human receptor for HDL (Murao et al. 1997, Imachi et al. 2003). Adipose tissue contains a very large pool of free cholesterol (Zhang et al. 2010). In fact, adipocytes are known to support in vitro cholesterol efflux to HDL and apoA-I (Prattes et al. 2000). Recent literature shows that SR-BI is expressed in mature adipocytes, and adipocyte cholesterol homeostasis may be regulated in a cell-specific manner (Prattes et al. 2000, Le Lay et al. 2003). SR-BI is a 509-amino acid glycoprotein with two cytoplasmic C-terminal and N-terminal domains, separated by a large extracellular domain. The extracellular domain of SR-BI is not only important in binding HDL particles but is also required for the efficient and selective cholesterol uptake of HDL-C ester (Linton et al. 2017). Additionally, the C-terminal tail of hSR-BI/CLA-1 might play an important role in HDL-mediated signal transduction mechanisms, including the PI3K/Akt signaling pathway and the calcium-mediated signal transduction pathway (Cao et al. 2004a , Linton et al. 2017).
However, the underlying mechanism that regulates APN expression via HDL receptor has yet to be revealed. In this study, we have examined the effect of HDL-hSR-BI/CLA-1 signal transduction mechanism on APN gene expression.
Materials and methods
Cell culture
The 3T3L1 cells originated from a mouse adipose cell line (American Type Culture Collection). The undifferentiated 3T3L1 cells were cultured in Dulbecco’s Modified Eagle’s Medium with 25 mM glucose (DMEM, Gibco-BRL, Tokyo, Japan) and supplemented with 10% newborn fetal bovine serum (FBS; Thermo Electron Co., Melbourne, Australia), 4 mM l-glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. To introduce differentiation, 3T3L1 cells were grown in DMEM supplemented with 10% FBS and the standard differentiation inducing mix: insulin (10 µg/mL, Sigma), dexamethasone (2.5 µM, Sigma), and isobutylmethylxanthine (IBMX, 0.5 mM, Sigma) for the first 2 days. For the next 2 days, the medium was supplemented only with 100 nM insulin. From day 5, the cells were switched back to plain DMEM with 10% FBS. The cells would be fully differentiated on day 10 and could be used until days 15–17. Y-1 and COS-7 cells (American Type Culture Collection) were grown in DMEM supplemented with 10% FBS. Cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C.
Western blot analysis
Cells were washed, scraped in PBS, and lysed as described previously (Murao et al. 2008). The proteins (10 μg) were separated on a 7.5% SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes for immunoblot assay. The membranes were incubated for 1 h at 4°C with 0.2% Tween 20 in PBS containing anti-APN antiserum (dilution, 1:250; Abcam) or anti-hSR-BI/CLA-1 antiserum (dilution, 1:2000) as described previously (Murao et al. 1997). The binding of each antibody was visualized using a chemiluminescence detection kit (enhanced chemiluminescence; Amersham Pharmacia Biotech). An antibody for GAPDH (diluted 1:1000; Trevigen, Gaithersburg, MD, USA) was used as the internal standard for the cytosolic extract. The membranes were then washed with PBS-T and incubated for 1 h at room temperature in PBS-T containing a second antibody linked to horseradish peroxidase. The signal was visualized using an enhanced chemiluminescence detection kit (ECL; GE Healthcare) under Luminescent image analyzer LAS-1000 Plus (Fujifilm). Band intensities were quantified using Multi Gauge V 3.2 software (Fujifilm).
Quantitative real-time PCR
Total RNA was extracted with RNA-Bee-RNA isolation reagent (Tel-Test Inc., Friendswood, TX, USA) from the mouse adipose and liver tissue, and RT was performed using Superscript II (Invitrogen) and 6 µg of total RNA; cDNA was used for quantitative real-time PCR (qPCR) using the Fast Start DNA Master SYBR Green I kit (Roche) in a CFX96 Real-time PCR Detection system (Bio-Rad) as previously described (Lyu et al. 2018). GAPDH was used as the housekeeping gene. The qPCR was performed using the following specific primer sequences:
GAPDH forward: 5’-TGAACGGGAAGCTCACTGG-3’ and reverse: 5’-TCCACCACCCTGTTGCTGTA-3’;
APN forward: 5’-AAGGACAAGGCCGTTCTC-3’ and reverse: 5’-AGAGTCGTTGACGTTATCTGCATAG-3’.
Transfection of small interfering RNA
The small interfering RNAs (siRNAs) designed to target SR-BI or scramble were purchased from Santa Cruz. Transfection of SR-BI siRNA was performed using siPORT Amine (Ambion) as described previously (Fukata et al. 2014).
Cholesterol content assay
For cholesterol content measurement, we employed a method developed by Shahnaz et al. (1998), which is a completely automated fluorimetric method for the determination of cellular cholesterol, consisting of enzymatic hydrolysis of cholesteryl ester to free cholesterol and enzymatic oxidation of free cholesterol in the presence of an indicator substrate to produce a fluorescent product.
Phosphorylation of Ca2+/calmodulin-dependent protein kinase IV at Thr196
3T3L1 cells were treated with 100 μg/mL HDL (Merck Millipore) for 2 min and harvested at predetermined time intervals. The cells were lysed, and endogenous Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK)IV was immunoprecipitated using the anti-CaMKIV antibody by the following procedure. Antibodies directed against CaMKIV (dilution, 1:2000; Abcam) were pre-incubated with protein G agarose for 30 min with rotation in 500 µL harvesting buffer and collected after centrifugation. Following aspiration, 1 mL of clarified cell extract was added to the immobilized antibody–protein G complex and tumbled overnight at 4°C. Following centrifugation, supernatants were removed and analyzed by western blotting. Western blotting analysis was carried out using anti-phospho-Thr196 antibody. The total cell lysate was also subjected to western blotting analysis using CaMKIV antibody as control. Anti-phospho-CaMKIV Thr196 monoclonal antibodies were generated against the synthetic phosphopeptides corresponding to residues 189–203 of rat CaMKIV (CEHQVLMKT(p)VCGTPGY) (Tokumitsu et al. 2004). Peptide was conjugated using keyhole limpet hemocyanin through the N-terminus cysteine and was injected into BALB/c mice as described previously (Kimura et al. 1994).
Transfection of 3T3L1 cells and luciferase reporter gene assay
To confirm the transcriptional regulation of the APN promoter by HDL, we used a plasmid construct containing the mouse APN promoter obtained by PCR amplification, cloned in front of the luciferase reporter gene as previously described (Li et al. 2010a). WT (−984 WT LUC) contains the mouse APN gene sequences spanning the region from −984 to +1, linked to the luciferase reporter gene (Li et al. 2010a). Purified reporter plasmid was co-transfected with a CaMKIV-expressing plasmid or an empty vector and a β-galactosidase expression plasmid (for determining transfection efficiency into the 3T3L1 cells at 70% confluence) using Lipofectamine (Life Technologies). Both cDNA of Ca2+/CaM-independent mutant of CaM-KIV (CaM-KIVc, 305 HMDT to DEDD) and CaM-KIV kinase-negative mutant (CaM-KIVdn, 305 HMDT to DEDD, K71E) were constructed as described previously (Kimura et al. 1994, Tokumitsu et al. 1994, Tokumitsu & Soderling 1996, Li et al. 2010a). Transfected cells were maintained in control media for 24 h as previously described (Lyu et al. 2020). Transfected cells were harvested, and β-galactosidase activity was measured in an aliquot of the cytoplasmic preparation. Twenty-microliter aliquots were taken for the luciferase assay, which was performed according to the manufacturer’s instructions (ToyoInk, Tokyo, Japan).
Statistical analysis
Data were expressed as mean ± s.e.m. Data were analyzed statistically using SPSS Statistics V17.0 software, and the significance of the differences between the corresponding groups was determined by ANOVA followed by one-tailed multiple t-tests. P value <0.05 was considered as statistically significant.
Results
Expression of SR-BI/CLA-1 in differentiated and undifferentiated 3T3-L1 cells
SR-BI/CLA-1 expression has been confirmed in mouse-derived Y-1 adrenal cells. Using Y-1 cells as a positive control, SR-BI/CLA-1 expression was examined in mouse-derived undifferentiated 3T3-L1 cells, differentiated 3T3-L1 cells, and mouse adipose tissue. As shown in Fig. 1, SR-BI/CLA-1 was expressed in mouse adipose tissue as well as in undifferentiated and differentiated 3T3-L1 cells.
Expression of SR-BI/CLA-1 in 3T3L1 cells. Total cell protein (10 µg) extracted from the adipose tissue and 3T3L1 cells was blotted with anti-SR-BI/CLA-1 or GAPDH antibody, respectively. Abundance of GAPDH served as a control. Lane 1, Y-1 cell; lane 2, mouse adipose tissue; lane 3, undifferentiated 3T3L1 cell; lane 4, differentiated 3T3L1 cell.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
Expression of SR-BI/CLA-1 in 3T3L1 cells. Total cell protein (10 µg) extracted from the adipose tissue and 3T3L1 cells was blotted with anti-SR-BI/CLA-1 or GAPDH antibody, respectively. Abundance of GAPDH served as a control. Lane 1, Y-1 cell; lane 2, mouse adipose tissue; lane 3, undifferentiated 3T3L1 cell; lane 4, differentiated 3T3L1 cell.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
Expression of SR-BI/CLA-1 in 3T3L1 cells. Total cell protein (10 µg) extracted from the adipose tissue and 3T3L1 cells was blotted with anti-SR-BI/CLA-1 or GAPDH antibody, respectively. Abundance of GAPDH served as a control. Lane 1, Y-1 cell; lane 2, mouse adipose tissue; lane 3, undifferentiated 3T3L1 cell; lane 4, differentiated 3T3L1 cell.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
Effect of HDL on adiponectin gene expression in 3T3-L1 cells
The effect of lipoproteins on APN expression in 3T3-L1 cells was examined by exposing the cells to 100 μg/mL of both LDL and HDL for 24 h. APN expression was examined by qPCR analysis to assess the level of mRNA transcription in the cells. The results showed an induction of the APN gene in response to HDL, but not LDL (Fig. 2A). We hypothesized that the transcriptional activity of the APN promoter is regulated by HDL in 3T3-L1 cells. In order to examine this hypothesis, we measured the APN promoter activity using the luciferase reporter gene assay in differentiated 3T3-L1 cells in the presence of HDL (Fig. 2B). HDL was found to have a stimulatory effect on the activity of the APN promoter. The results showed that HDL treatment stimulated the promoter activity of APN.
(A) HDL-stimulated APN gene expression. The effect of HDL on mRNA expression of APN by real-time PCR methods as described in the methods section. The ratio is shown as percent of control. A graph showing the mean ± s.e.m. (n = 3) of separate experiments for each treatment group is indicated. (B) HDL-stimulated APN promotor activity. 3T3L1 cells were transfected with 1 μg of pAPN-LUC and treated with 100 μg/mL HDL for 24 h prior to cell harvesting. All assays were corrected for β-galactosidase activity, and the total amount of protein in every reaction was identical. The results were expressed as relative luciferase activity compared with the control cells arbitrarily set at 100. Each data point shows the mean ± s.e. of three separate transfections that were performed on separate days. *P < 0.05 compared to control. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
(A) HDL-stimulated APN gene expression. The effect of HDL on mRNA expression of APN by real-time PCR methods as described in the methods section. The ratio is shown as percent of control. A graph showing the mean ± s.e.m. (n = 3) of separate experiments for each treatment group is indicated. (B) HDL-stimulated APN promotor activity. 3T3L1 cells were transfected with 1 μg of pAPN-LUC and treated with 100 μg/mL HDL for 24 h prior to cell harvesting. All assays were corrected for β-galactosidase activity, and the total amount of protein in every reaction was identical. The results were expressed as relative luciferase activity compared with the control cells arbitrarily set at 100. Each data point shows the mean ± s.e. of three separate transfections that were performed on separate days. *P < 0.05 compared to control. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
(A) HDL-stimulated APN gene expression. The effect of HDL on mRNA expression of APN by real-time PCR methods as described in the methods section. The ratio is shown as percent of control. A graph showing the mean ± s.e.m. (n = 3) of separate experiments for each treatment group is indicated. (B) HDL-stimulated APN promotor activity. 3T3L1 cells were transfected with 1 μg of pAPN-LUC and treated with 100 μg/mL HDL for 24 h prior to cell harvesting. All assays were corrected for β-galactosidase activity, and the total amount of protein in every reaction was identical. The results were expressed as relative luciferase activity compared with the control cells arbitrarily set at 100. Each data point shows the mean ± s.e. of three separate transfections that were performed on separate days. *P < 0.05 compared to control. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
HDL increases adiponectin promoter activity in 3T3-L1 cells via the CaMKK pathway
To test whether the interaction of HDL and its membrane receptor is involved in a signaling pathway, we used known inhibitors of signaling pathways to disrupt HDL induction of ANP promoter activity. Thus, we treated pANP-LUC-transfected 3T3-L1 cells with compounds known to inhibit PI3-K (LY294002, 10 μmol/L), extracellular-signal-regulated kinase (PD98059, 10 μmol/L), protein kinase C (PKC) (bisindolylmaleimide I, 1 μmol/L), and p38-mitogen-activated protein kinase (p38-MAPK) (SB203580, 1 μmol/L) before exposing them to HDL. The results showed that all aforementioned inhibitors had no effect on the action of HDL; however, the STO-609 (1 µM), inhibitor for Ca2+/calmodulin (CaM)-dependent protein kinase kinase (CaMKK2), abrogated the ability of HDL to induce APN promoter activity (Fig. 3A). The STO-609 treatment therefore also inhibited the expression of APN (Fig. 3B). These results suggest that the effects of HDL are mediated via the CaMKK cascade. It is well known that HDL promotes cholesterol efflux from cells. Therefore, we investigated the effect of STO-609 on cholesterol efflux in HDL-treated 3T3L1 cells. To assess the effect of STO-609 on cholesterol efflux, cholesterol content in HDL-treated 3T3L1 cells was measured with or without STO-609 treatment. The results showed that the addition of STO-609 increased the cholesterol content of 3T3L1 cells (Supplemental data). This showed that STO-609 inhibited the efflux of cholesterol from cells.
(A) STO-609, inhibitor for Ca2+/calmodulin (CaM)-dependent protein kinase kinase (CaMKK2), abrogates the effect of HDL on APN expression. The effect of STO-609 on HDL-induced APN transcriptional activity. Cont; without STO-609, STO-609; with STO-609 (1 µM). All assays were corrected for β-galactosidase activity, and the total amount of protein in every reaction was identical. The results were expressed as relative luciferase activity compared with the control cells arbitrarily set at 100. Each data point shows the mean ±s.e.of four separate transfections that were performed on separate days. (B) STO-609, inhibitor for CaMKK2, abrogates the effect of HDL on APN expression. The effect of STO-609 on HDL-induced APN mRNA expression by real-time PCR methods. The ratio is shown as percentage of control. A graph showing the mean ± s.e.m. (n = 3) of separate experiments for each treatment group is indicated. *P < 0.05 compared to control. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests. N.S., no significance.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
(A) STO-609, inhibitor for Ca2+/calmodulin (CaM)-dependent protein kinase kinase (CaMKK2), abrogates the effect of HDL on APN expression. The effect of STO-609 on HDL-induced APN transcriptional activity. Cont; without STO-609, STO-609; with STO-609 (1 µM). All assays were corrected for β-galactosidase activity, and the total amount of protein in every reaction was identical. The results were expressed as relative luciferase activity compared with the control cells arbitrarily set at 100. Each data point shows the mean ±s.e.of four separate transfections that were performed on separate days. (B) STO-609, inhibitor for CaMKK2, abrogates the effect of HDL on APN expression. The effect of STO-609 on HDL-induced APN mRNA expression by real-time PCR methods. The ratio is shown as percentage of control. A graph showing the mean ± s.e.m. (n = 3) of separate experiments for each treatment group is indicated. *P < 0.05 compared to control. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests. N.S., no significance.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
(A) STO-609, inhibitor for Ca2+/calmodulin (CaM)-dependent protein kinase kinase (CaMKK2), abrogates the effect of HDL on APN expression. The effect of STO-609 on HDL-induced APN transcriptional activity. Cont; without STO-609, STO-609; with STO-609 (1 µM). All assays were corrected for β-galactosidase activity, and the total amount of protein in every reaction was identical. The results were expressed as relative luciferase activity compared with the control cells arbitrarily set at 100. Each data point shows the mean ±s.e.of four separate transfections that were performed on separate days. (B) STO-609, inhibitor for CaMKK2, abrogates the effect of HDL on APN expression. The effect of STO-609 on HDL-induced APN mRNA expression by real-time PCR methods. The ratio is shown as percentage of control. A graph showing the mean ± s.e.m. (n = 3) of separate experiments for each treatment group is indicated. *P < 0.05 compared to control. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests. N.S., no significance.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
CaMKK/CaMKIV cascade mediates HDL-induced ANP gene expression
Phosphorylation of CaMKIV by HDL in 3T3-L1 cells
Our study indicated that the CaMKK pathway might be involved in the enhancement of HDL-mediated APN gene expression in 3T3-L1 cells. It has been reported that CaMKK is capable of activating multiple protein kinases including CaMKI, CaMKIV, PKB, AMP-kinase, and SAD-B kinase. To determine which downstream effector kinase of CaMKK is involved in this mechanism, we first examined the ability of HDL to stimulate CaMKIV activity in 3T3-L1 cells, since CaMKIV has shown to be localized in nucleus and involved in the transcription, through the phosphorylation of transcriptional factors such as cAMP response element-binding (CREB) protein. We confirmed that CaMKK/CaMKIV was expressed in 3T3-L1 cells. As shown in Fig. 4A, it was found that CaMKK/CaMKIV was expressed in differentiated 3T3-L1 cells in the same manner as the positive control, in INS-1 cells. Previous studies showed that the phosphorylation of Thr196 residues in the activation loop of CaMKIV by CaMKK greatly induced its kinase activity. Therefore, we examined Thr196 phosphorylation of CaMKIV in response to HDL in 3T3-L1 cells. 3T3-L1 cells were treated with HDL and harvested at predetermined time intervals. Cells were then analyzed using western blotting with either anti-phospho-Thr196 antibodies (Fig. 4B, upper panel) or anti-CaMKIV antibodies (Fig. 4B, lower panel). 3T3-L1 cells treated with HDL showed an increase in CaMKIV phosphorylation at Thr196. The upregulation of CaMKIV phosphorylation peaked after 15 min of HDL treatment.
The role of CaMKK/CaMKIV pathway on HDL-induced APN expression. (A) The expression of CaMKK2 or CaMKIV in 3T3L1 cells. Total cell protein (10 µg) extracted from 3T3L1 cells or INS-1 cells was blotted with anti-CaMKK2 or anti-CaMKIV antibody, respectively. Abundance of GAPDH served as a control. COS-7 (control), INS-1 (positive control), undi3T3L1 (undifferentiated 3T3L1 cells), di3T3L1 (differentiated 3T3L1 cells). (B) Phosphorylation of CaMKIV by HDL in 3T3L1 cells. Post-confluent 3T3-L1 preadipocytes treated with differentiation medium, which contains a mixture of IBMX, dexamethasone, and insulin, initiated adipogenic differentiation and intracellular lipid accumulation was marked by day 10, suggesting that 3T3-L1 preadipocytes differentiated into mature adipocytes. 3T3L1 cells were exposed to 100 μg/mL HDL for 2 min before harvest at the predetermined time interval. The total cell extracts (10 µg) were subjected to immunoprecipitation using anti-CaMKIV antibodies and SDS-PAGE, followed by western blotting analysis using anti-phospho-Thr196 antibodies (upper panel). Total cell lysates (10 µg) were also blotted using anti-CaMKIV antibodies as a control (lower panel). An identical experiment independently performed yielded similar results. (C) Effect of the CaMKIV on APN promoter activity. The cells were transfected with APN-LUC and empty vector (pcDNA3) or CaMKIVc expression vectors. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e.m. (n = 3) of separate transfections. The asterisk denotes the significant difference (P < 0.05). (D) CaMKIV-dominant negative (CaMKIVdn) mutant inhibits the upregulation of APN promoter activity by HDL. 3T3L1 cells were transfected with APN-LUC and CaMKIVdn for 24 h prior to measuring luciferase activity. Cont, APN-LUC and pcDNA3, CaMKIVdn; APN-LUC and CaMKIVdn expression vector with or without HDL. The results are expressed as relative luciferase activities compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e.m. (n = 3) of separate transfections. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
The role of CaMKK/CaMKIV pathway on HDL-induced APN expression. (A) The expression of CaMKK2 or CaMKIV in 3T3L1 cells. Total cell protein (10 µg) extracted from 3T3L1 cells or INS-1 cells was blotted with anti-CaMKK2 or anti-CaMKIV antibody, respectively. Abundance of GAPDH served as a control. COS-7 (control), INS-1 (positive control), undi3T3L1 (undifferentiated 3T3L1 cells), di3T3L1 (differentiated 3T3L1 cells). (B) Phosphorylation of CaMKIV by HDL in 3T3L1 cells. Post-confluent 3T3-L1 preadipocytes treated with differentiation medium, which contains a mixture of IBMX, dexamethasone, and insulin, initiated adipogenic differentiation and intracellular lipid accumulation was marked by day 10, suggesting that 3T3-L1 preadipocytes differentiated into mature adipocytes. 3T3L1 cells were exposed to 100 μg/mL HDL for 2 min before harvest at the predetermined time interval. The total cell extracts (10 µg) were subjected to immunoprecipitation using anti-CaMKIV antibodies and SDS-PAGE, followed by western blotting analysis using anti-phospho-Thr196 antibodies (upper panel). Total cell lysates (10 µg) were also blotted using anti-CaMKIV antibodies as a control (lower panel). An identical experiment independently performed yielded similar results. (C) Effect of the CaMKIV on APN promoter activity. The cells were transfected with APN-LUC and empty vector (pcDNA3) or CaMKIVc expression vectors. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e.m. (n = 3) of separate transfections. The asterisk denotes the significant difference (P < 0.05). (D) CaMKIV-dominant negative (CaMKIVdn) mutant inhibits the upregulation of APN promoter activity by HDL. 3T3L1 cells were transfected with APN-LUC and CaMKIVdn for 24 h prior to measuring luciferase activity. Cont, APN-LUC and pcDNA3, CaMKIVdn; APN-LUC and CaMKIVdn expression vector with or without HDL. The results are expressed as relative luciferase activities compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e.m. (n = 3) of separate transfections. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
The role of CaMKK/CaMKIV pathway on HDL-induced APN expression. (A) The expression of CaMKK2 or CaMKIV in 3T3L1 cells. Total cell protein (10 µg) extracted from 3T3L1 cells or INS-1 cells was blotted with anti-CaMKK2 or anti-CaMKIV antibody, respectively. Abundance of GAPDH served as a control. COS-7 (control), INS-1 (positive control), undi3T3L1 (undifferentiated 3T3L1 cells), di3T3L1 (differentiated 3T3L1 cells). (B) Phosphorylation of CaMKIV by HDL in 3T3L1 cells. Post-confluent 3T3-L1 preadipocytes treated with differentiation medium, which contains a mixture of IBMX, dexamethasone, and insulin, initiated adipogenic differentiation and intracellular lipid accumulation was marked by day 10, suggesting that 3T3-L1 preadipocytes differentiated into mature adipocytes. 3T3L1 cells were exposed to 100 μg/mL HDL for 2 min before harvest at the predetermined time interval. The total cell extracts (10 µg) were subjected to immunoprecipitation using anti-CaMKIV antibodies and SDS-PAGE, followed by western blotting analysis using anti-phospho-Thr196 antibodies (upper panel). Total cell lysates (10 µg) were also blotted using anti-CaMKIV antibodies as a control (lower panel). An identical experiment independently performed yielded similar results. (C) Effect of the CaMKIV on APN promoter activity. The cells were transfected with APN-LUC and empty vector (pcDNA3) or CaMKIVc expression vectors. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e.m. (n = 3) of separate transfections. The asterisk denotes the significant difference (P < 0.05). (D) CaMKIV-dominant negative (CaMKIVdn) mutant inhibits the upregulation of APN promoter activity by HDL. 3T3L1 cells were transfected with APN-LUC and CaMKIVdn for 24 h prior to measuring luciferase activity. Cont, APN-LUC and pcDNA3, CaMKIVdn; APN-LUC and CaMKIVdn expression vector with or without HDL. The results are expressed as relative luciferase activities compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e.m. (n = 3) of separate transfections. The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
Role of CaMKIV in HDL-induced APN gene expression
The results of this experiment so far identified the components of the CaMKK cascade in HDL-induced APN promoter activity. The lack of information on whether this signal transduction cascade mediates the action of HDL in 3T3-L1 cells led us to examine whether CaMKIV plays a role in the HDL-induced transcription of the APN gene. Therefore, we examined the potential role of CaMKIV by co-transfecting 3T3-L1 cells with the APN promoter gene and CaMKIV to determine whether CaMKIV affected APN gene transcription. Results showed that CaMKIVc stimulated APN promoter activity in 3T3-L1 cells (Fig. 4C). Furthermore, when 3T3-L1 cells were co-transfected with a dominant negative mutant of CaMKIV (CaMKIVdn), the increase in HDL-induced APN promoter activity was entirely suppressed (Fig. 4D). Together, these findings support the notion that the CaMKK/CaMKIV cascade is required for APN expression induced by HDL in 3T3-L1 cells.
Role of SR-BI/CLA-1 in HDL-induced APN expression
To further characterize the role of SR-BI/CLA-1 in the HDL-mediated signaling that enhances APN expression, we used siRNA to block SR-BI/CLA-1-expression. 3T3-L1 cells were exposed to SR-BI/CLA-1-specific siRNA and scramble siRNA and then treated with HDL. As shown in Fig. 5, APN protein expression was increased in cells exposed to scrambled siRNA following stimulation with HDL. In contrast, the increase in HDL-induced APN expression was significantly suppressed in cells treated with SR-BI-siRNA. The above-mentioned findings support the idea that SR-BI/CLA-1 plays a role in HDL-induced APN expression.
Effects of SR-BI/CLA-1-knockdown on APN expression by HDL. SR-BI/CLA-1 (siRNA-SR-BI/CLA-1) siRNA or scrambled siRNA (siRNA-Cont) was transfected into 3T3L1 cells with or without HDL. The upper panel shows that the abundance of APN protein level was measured using western blot analysis at 48 h after transfection. The ratio of APN to GAPDH is represented as a percentage of the control. Each data point shows the mean ± s.e.m. (n = 3) of separate experiments. The asterisk denotes a significant difference (P < 0.05). The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests. N.S., no significance.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
Effects of SR-BI/CLA-1-knockdown on APN expression by HDL. SR-BI/CLA-1 (siRNA-SR-BI/CLA-1) siRNA or scrambled siRNA (siRNA-Cont) was transfected into 3T3L1 cells with or without HDL. The upper panel shows that the abundance of APN protein level was measured using western blot analysis at 48 h after transfection. The ratio of APN to GAPDH is represented as a percentage of the control. Each data point shows the mean ± s.e.m. (n = 3) of separate experiments. The asterisk denotes a significant difference (P < 0.05). The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests. N.S., no significance.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
Effects of SR-BI/CLA-1-knockdown on APN expression by HDL. SR-BI/CLA-1 (siRNA-SR-BI/CLA-1) siRNA or scrambled siRNA (siRNA-Cont) was transfected into 3T3L1 cells with or without HDL. The upper panel shows that the abundance of APN protein level was measured using western blot analysis at 48 h after transfection. The ratio of APN to GAPDH is represented as a percentage of the control. Each data point shows the mean ± s.e.m. (n = 3) of separate experiments. The asterisk denotes a significant difference (P < 0.05). The differences between the corresponding groups were determined by ANOVA followed by one-tailed multiple t-tests. N.S., no significance.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-20-0211
Discussion
In this report, we have summarized the results of our studies, showing that HDL regulated APN gene expression via hSR-BI/CLA-1. Furthermore, we have explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. This effect appears to be mediated by CaMKK due to a specific inhibitor of this kinase, STO-609, which prevents HDL-stimulated APN expression. To further define the actions of HDL, we used a transient transfection assay to clarify the signal transduction pathway that mediated HDL-induced APN gene expression. We revealed that CaMKIVc increased APN gene transcriptional activity. Finally, knockdown of hSR-BI/CLA-1 canceled the effect of HDL on APN gene expression. In this study, our results suggest that HDL improves the function of adipocytes by activating hSR-BI/CLA-1, and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.
The cellular uptake of cholesterol in HDL is facilitated by SR-BI. This protein was identified as an HDL receptor in rodents (Acton et al. 1996). Human CD36 and LIMPII Analogous-1 (CLA-1) share 81% sequence homology with hamster SR-BI (Calvo & Vega 1993). Our previous reports show that human SR-BI (hSR-BI/CLA-1), like mouse SR-BI, functions as a receptor for HDL (Murao et al. 1997, Imachi et al. 1999, 2003, Cao et al. 2004a,b). Human SR-BI/CLA-1 is also similar to the mouse homolog because it can mediate selective uptake of cholesterol ester and is expressed in the liver and steroidogenic tissues. Several reports indicated that SR-BI is expressed in adipocytes and adipose tissues (Zhang et al. 2010). Adipose tissues harbor a large depot of free cholesterol. HDL has an important role to remove excess cholesterol from peripheral tissues and cells. The process of peripheral cholesterol efflux to HDL and delivery to the liver was also observed in adipocytes (Wu & Zhao 2009, Mostafa et al. 2015). It was revealed that adipocytes support the transfer of cholesterol to HDL in vivo as well as in vitro and implicate ABCA1 and SR-BI cholesterol transporters (Zhao et al. 2006). However, the role of SR-BI in adipocytes has not yet been fully investigated. Although SR-BI is the receptor that mediates cholesterol transport in HDL metabolism, in this report we have revealed that it may be the sensing receptor for HDL-dependent signal transduction.
In endothelial cells, a series of studies have established that SR-BI interaction with HDL prevents endothelial dysfunction by stimulating eNOS activity (Mineo & Shaul 2013). Regarding its mechanisms, we previously reported that the C-terminal tail of hSR-BI/CLA-1 might play an important role in HDL-mediated signal transduction mechanisms, including the PI3K/Akt signaling pathway (Yu et al. 2007). The PDZ domain-containing protein 1 (PDZK1), also known as CLAMP, was first identified as an SR-BI-associated protein in affinity chromatography studies of rat liver membrane extracts (Kocher & Krieger 2009). PDZK1 contains four PDZ domains. The N-terminal domain interacts with the extreme C-terminal residues of SR-BI and PDZK1, which is linked to the PI3K/Akt signaling pathway. It was previously reported that increased HDL-C following human apoA-I transfer affects the expression of genes involved in fatty acid and triglyceride metabolism in adipose tissue. It was also reported that increased HDL-C potently attenuates the increase of triglycerides and free fatty acids induced by LPS administration, whereas HDL elevates plasma APN concentrations in vivo and increases APN expression in adipocytes in a PI3K-dependent manner (Van Linthout et al. 2010).
In addition to the previously mentioned signaling pathways activated by HDL via SR-BI, other signaling events have also been reported in HDL-treated cells. These include the activation of phosphatidylinositol and phosphatidylcholine-specific phospholipases (PI-PLC, PC-PLC, and PC-PLD), protein kinase C (PKC), heterotrimeric G proteins, the production of 3’,5’-cAMP and ceramide, and the stimulation of intracellular calcium release (Saddar et al. 2010). The importance of calcium signaling in HDL-stimulated gene expression was reported previously (Romero et al. 2006). Preincubation with nifedipine, a calcium channel blocker, completely blocked HDL stimulation of CYP11B2 in adrenal cells, whereas KN93, a calmodulin kinase inhibitor, inhibited 80% of the stimulation.
Our study identified the role of the CaMKK/CaMKIV cascade in APN gene expression in response to HDL. Numerous studies have demonstrated that the CaMKK/CaMKIV cascade is present and functional in various cell types, including pancreatic β-cells (Li et al. 2010b, Chen et al. 2011). We previously reported that pancreatic β-cells incorporate the CaMKK/CaMKIV cascade and that it plays an important role in glucose-upregulated transcriptional activation of the insulin gene (Yu et al. 2004). In this study, we confirmed that this pathway also exists in adipocytes, and HDL induced the phosphorylation of CaMKIV at Thr196 by CaMKK. We also confirmed that the expression of CaMKIVdn canceled the effect of HDL on HDL-induced APN expression, raising the possibility that activated CaMKIV mediates the stimulatory effect of HDL-dependent APN gene expression. Several studies have reported that APN is transcriptionally regulated by various transcription factors, such as peroxisome proliferator-activated receptor γ (PPARG, also known as PPARγ; (Iwaki et al. 2003)), CCAAT/enhancer-binding protein-α (CEBPA, also known as C/EBPα; (Park et al. 2004)), nuclear factor of activated T cells 4 (NFAT4), and activating transcription factor three (ATF3; (Kim et al. 2006)) through cis-regulatory elements in its proximal promoter region. The active, phosphorylated form of CaMKIV has been shown to translocate to the nucleus, where it regulates the transcriptional activity of several targets that play crucial roles in immune response, inflammation, and cellular proliferation (Chow et al. 2005). Activation of CaMKIV has been associated with increased phosphorylation of CREB, NFAT, and serum response factor protein (Jensen et al. 1991). The APN promoter region has the binding sites for these transcription factors. Further studies are needed to investigate the mechanism of CaMKIV-mediated transcriptional activation of the APN gene.
Of the reviewed studies, almost all reported that circulating APN is positively correlated with the serum level of HDL-C (Christou & Kiortsis 2013). HDL-C has also been shown to be positively correlated with HMW APN. Low levels of APN are associated with type 2 diabetes, NASH, and atherosclerosis (Phillips & Kung 2010). Although the underlying mechanisms responsible for hypoadiponectinemia in obesity have not been discovered, increased fat deposits result in adipose tissue hypoxia, thereby increasing endoplasmic reticular stress. Obesity also induces macrophage filtration into adipocytes, resulting in a low-grade chronic inflammatory state accompanied by an increase in the production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-18 (Su et al. 2011). These factors that induce hypoadiponectinemia may also be involved in the onset of type 2 diabetes. Previously, we reported that the hyperglycemic state inhibits the expression of SR-BI/CLA-1 via the p38MAPK-Sp1 pathway (Murao et al. 2008). Hyperglycemia is also related to hypoadiponectinemia, raising the possibility that a lower expression of SR-BI/CLA-1 suppresses HDL-dependent APN gene expression in adipocytes. Clinical studies suggest that hypoadiponectinemia contributes to insulin resistance, and elevated serum APN levels are associated with increased insulin sensitivity and reduced cardiovascular injury (Kadowaki et al. 2006). Circulating APN levels are increased by many commonly used drugs, such as statins, thiazolidinediones (TZDs), and angiotensin-converting enzyme inhibitors (Su et al. 2011). We previously reported that angiotensin II (Ang II) inhibited the expression of SR-BI/CLA-1 via PI3K/Akt/FoxO1 pathway, and angiotensin II receptor type 1 antagonist rescued the effect of Ang II, resulting in the upregulation of SR-BI/CLA-1. The upregulation of Ang II-inhibited hSR-BI/CLA-1 expression might represent a novel mechanism contributing to the anti-hypoadiponectinemia of AT type 1 receptor blockade in adipose tissue (Yu et al. 2007). Further investigation is needed to determine the detailed mechanisms involved in the regulation of APN gene by the HDL-SR-BI/CLA-1 pathway.
In summary, we examined the role of the CaMKK/CaMKIV cascade in HDL-induced APN gene expression via SR-BI/CLA-1. The results indicated that HDL has an important role to improve the function of adipocytes by activating hSR-BI/CLA-1, and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism, but we could not fully explain about direct connection between CaMKK/CaMKIV pathway and hSR-BI/CLA-1. Further consideration might be needed in the future research.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JME-20-0211.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was funded in part by Grant-in-Aid for the Ministry of Education, Culture, Sports, Science and Technology, Japan (18K08518) to K M, Grant-in-Aid for Research activity start-up (19K23970) to S S and Grant-in-Aid for Early-Career Scientists (20K17513) to J L.
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