Abstract
Prominin-1 (Prom1) is a major cell surface marker of cancer stem cells, but its physiological functions in the liver have not been elucidated. We analyzed the levels of mRNA transcripts in serum-starved primary Prom1+/+ and Prom1-/- mouse hepatocytes using RNA-sequencing (RNA-seq) data, and found that CREB target genes were down-regulated. This initial observation led us to determine that the Prom1 deficiency inhibited cAMP response element binding protein (CREB) activation and gluconeogenesis, but not cyclic AMP (cAMP) accumulation, in glucagon-, epinephrine-, or forskolin-treated liver tissues and primary hepatocytes, and mitigated glucagon-induced hyperglycemia. Because Prom1 interacted with radixin, the Prom1 deficiency prevented radixin from localizing to the plasma membrane. Moreover, systemic adenoviral knockdown of radixin inhibited CREB activation and gluconeogenesis in glucagon-treated liver tissues and primary hepatocytes, and mitigated glucagon-elicited hyperglycemia. Based on these results, we conclude that Prom1 regulates hepatic PKA signaling via radixin functioning as an A kinase-anchored protein (AKAP).
Introduction
In well understood glucagon-elicited signaling pathway glucagon binds to and stimulates glucagon receptor, a G-protein-coupled receptor, in the plasma membrane of hepatocytes. The stimulated glucagon receptor in turn activates Gsα and adenylyl cyclase (AC). The cyclic AMP (cAMP) produced by the activated AC binds to the regulatory domain of protein kinase A (PKA) to liberate the catalytic domain of PKA from the regulatory domain and activate the protein. Not only does activated PKA induces glycogenolysis by consecutively phosphorylating and activating phosphorylase kinase and glycogen phosphorylase b, but also stimulates gluconeogenesis by phosphorylating and activating cAMP response element binding protein (CREB). Subsequently, activated CREB induces the transcriptional activation of gluconeogenesis-related genes such as phosphoenolpyruvate carboxykinase (Pck) and glucose-6-phosphatase (G6p) (Altarejos & Montminy, 2011, Meinkoth, Alberts et al., 1993).
Detergent-resistant lipid rafts, which are composed of cholesterol and glycolipids and serve as a signaling center, facilitate the cascade of glucagon signaling by organizing glucagon receptor, Gsα, AC, A kinase-anchored proteins (AKAPs), PKA and PKA substrates in close proximity (Delint-Ramirez, Willoughby et al., 2011, Head, Patel et al., 2006, Kim, Lee et al., 2010). AKAPs are scaffolding proteins that bind PKA and its substrates. Thus, AKAPs allow PKA and its substrates to coexist in the same place, such as the plasma membrane, mitochondria or nucleus, enabling effective signal transduction by cAMP (Dema, Perets et al., 2015, Wong & Scott, 2004).
The penta-transmembrane glycoprotein prominin-1, also called CD133, localizes in membrane protrusions such as microvilli, filopodia and primary cilia in epithelial cells, membrane expansions in the myelin sheath originating from oligodendrocytes, membrane invaginations in the outer segment of rod photoreceptor cells and the midbody in epithelial cells (Corbeil, Roper et al., 2001, Corbeil, Roper et al., 1999, Corbeil, Roper et al., 2000, Dubreuil, Marzesco et al., 2007, Zacchigna, Oh et al., 2009). Mice with a systemic deficiency in Prom1 exhibit disk dysmorphogenesis and photoreceptor degeneration along with a complete loss of vision, indicating that Prom1 might be necessary for the formation of membrane extrusions (Zacchigna et al., 2009). In addition, Prom1 has been extensively studied as a major cancer stem cell marker in human brain, colon, ovarian and liver tumors. The Prom1-positive cell population in these tumors has characteristics of self-renewal, differentiation potential and resistance to chemo- and/or radiotherapy as well as tumor development after xenograft transplantation in immunocompromised mice (Dalerba, Dylla et al., 2007, Krishnan, Ochoa-Alvarez et al., 2013, Li, Heidt et al., 2007). Because PI3K interacts with Prom1 and Akt is highly phosphorylated in Prom1-positive cell populations, the Prom1-PI3K-Akt signaling pathway may be required for the maintenance of cancer stem cells (Wei, Jiang et al., 2013). However, this pathway has not been verified in a Prom1-deficient animal model.
Although Prom1 is expressed at high levels in various epithelial cells in the brain, kidney, digestive track and liver, the physiological functions of Prom1 are poorly understood. We analyzed glucagon-elicited gluconeogenesis in the livers of Prom1-deficient mice to understand Prom1 function. Here, we report that Prom1 is required for glucagon-induced PKA activation and hyperglycemia through its interaction with radixin, which functions as an AKAP in the liver.
Results
Prom1 is required for glucagon- and cAMP-elicited gluconeogenesis in mouse primary hepatocytes
After confirming Prom1 expression in mouse liver as well as primary hepatocytes (Fig S1A), we analyzed the levels of mRNA transcripts in serum-starved primary Prom1+/+ and Prom1-/- mouse hepatocytes using RNA-sequencing (RNA-seq) data to evaluate the transcriptional effects of Prom1 deficiency in the liver. We focused on the genes that were induced (n = 55) or repressed (n = 63) more than 2-fold with p < 0.05 (Fig 1A). Singular enrichment analysis of those 118 differentially expressed genes (DEG) for KEGG pathways revealed that pathways such as glycolysis/gluconeogenesis, glucagon and cAMP signaling pathway were highly enriched in the DEGs by Prom1 deficiency (Fig EV1B). Because the liver plays a central role in the regulation of glucose homeostasis, we focused on glycolysis/gluconeogenesis. Identified genes in these pathways included G6pc (glucose-6-phosphatase catalytic subunit), Pck1 (phosphoenolpyruvate carboxykinase 1), Pfkl (phosphofructokinase), and Pkm (pyruvate kinase), and were consistently downregulated by Prom1 deficiency (Fig 1A). These observations were further confirmed by qRT-PCR (Fig 1B), immunoblotting (Fig 1C) and glucose uptake assays (Fig 1D), and the results led us to suspect that Prom1 may be involved in hepatic gluconeogenesis.
To confirm the initial observations in mouse primary hepatocytes, we examined livers of Prom1+/+ and Prom1-/- mice in the fasting state. Expression of G6pc and Pck1 were decreased in Prom1-/- mouse liver compared to the level in Prom1+/+ mouse liver in the fasting state (Fig 1E). Fasting-induced CREB phosphorylation was also decreased in Prom1-/- mouse liver (Fig 1F). Prom1-/- mouse had lower blood glucose level without difference in blood glucagon levels (Fig 1G and H). Hepatic glycogen breakdown during fasting was reduced in Prom1-/- mice (Fig 1I). Livers of two groups did not show any histological difference (Fig S1C). These results suggested that loss of Prom1 interfered with the activation of hepatic gluconeogenesis.
To further confirm the involvement of Prom1 in hepatic gluconeogenesis, we determined the expression levels of hepatic gluconeogenic genes in Prom1+/+ and Prom1-/-mice hepatocytes after glucagon stimulation. The Prom1 deficiency interfered with the upregulation of G6pc and Pck1 expression in glucagon-stimulated primary hepatocytes (Fig 2A and Fig S1D). Because glucagon induces the upregulation of gluconeogenic genes by activation of CREB, we measured glucagon-induced CREB phosphorylation. In Prom1-deficient hepatocyte, glucagon-induced CREB phosphorylation was significantly decreased (Fig 2B) and nuclear localization of CREB was also decreased as a result (Fig 2C and Fig S1E).
The decreased CREB phosphorylation and the reduced upregulation of gluconeogenic genes in glucagon-induced Prom1-/- mouse hepatocytes led us to examine the effect of the Prom1 deficiency on glucagon receptor signaling pathway. The Prom1 deficiency reduced glucagon-induced phosphorylation of PKA substrates (Fig 2B and D). The PKA activity and cAMP level in the presence or absence of IBMX (3-isobutyl-1-methylxanthine), a phosphodiesterase (PDE) inhibitor, were measured in glucagon-stimulated Prom1+/+ and Prom1-/- primary hepatocytes to investigate the abilities of the AC and PDE enzymes required to produce and degrade cAMP, respectively. Blocking cAMP degradation by IBMX failed to restore PKA activity in Prom1-/- mouse hepatocytes (Fig 2D). The Prom1 deficiency did not change glucagon-induced cAMP production or degradation (Fig 2E). We also examined the effect of Prom1 overexpression on glucagon-induced CREB phosphorylation and cAMP production in Prom1-/- primary hepatocytes. Adenoviral Prom1 overexpression restored glucagon-induced CREB phosphorylation (Fig S1F), but did not affect glucagon-elicited cAMP production (Fig S1G).
To corroborate our observations, we tested two different PKA activators, forskolin (an AC activator) and 8-Br-cAMP (a non-degradable cAMP analogue) in Prom1+/+ and Prom1-/- primary hepatocytes. Consistent with the previous results, the Prom1 deficiency prevented forskolin- and 8-Br-cAMP-induced CREB phosphorylation (Fig S2A and B) and reduced glucagon-induced phosphorylation of PKA substrates even in the presence of IBMX (Fig S2C) without changing glucagon-induced cAMP production (Fig S2D). Taken together, we concluded that Prom1 regulated cAMP-induced PKA activation in primary hepatocytes.
Prom1 is necessary for glucagon-elicited hepatic gluconeogenesis in vivo
The requirement for Prom1 in glucagon-elicited gluconeogenesis was further analyzed in vivo by treating Prom1+/+ and Prom1-/- mice with glucagon. We conducted glucagon challenge tests in 12-week-old mice. Glucagon-elicited hyperglycemia was mitigated in Prom1-/- mice compared to that in Prom1+/+ mice (Fig 2F). The livers of Prom1-deficient mice also exhibited reduced glucagon-induced phosphorylation of CREB and PKA substrates (Fig 2G and H). However, Prom1-deficient mice did not display a change in glucagon-induced cAMP production in the liver (Fig S2E). Increased glucose internalization in Prom1-/- mouse hepatocytes (Fig 1D) led us to hypothesize that Prom1-deficient mice would show decreased blood glucose level due to the reduced hepatic gluconeogenic capacity. To test this hypothesis, we performed glucose, pyruvate and insulin tolerance tests in Prom1+/+ and Prom1-/- mice. Prom1-deficient mice displayed improved glucose and pyruvate tolerance (Fig 2I). Prom1 deficiency did not change insulin tolerance (Fig 2J), as insulin-induced signaling was not affected (Fig 2K). These results demonstrated that the Prom1deficiency prevented hepatic gluconeogenesis but did not affect insulin signaling.
Prom1 regulates β-adrenergic receptor signaling in vivo
Because glucagon receptor is a member of the GPCR (G-protein coupled receptor) family, we hypothesized that Prom1 would also regulate other GPCR signaling that involves PKA activation. To test this hypothesis we investigated PKA signaling in Prom1+/+ and Prom1-/- primary hepatocytes treated with isoprenaline, a β-adrenergic receptor agonist. The Prom1 deficiency prevented phosphorylation of CREB and PKA substrates (Fig 3A and B), but not cAMP production (Fig 3C) in isoprenaline-treated primary hepatocytes. To further validate that Prom1 regulates β-adrenergic receptor signaling, we examined blood glucose level and PKA activation in Prom1+/+ and Prom1-/- mice after epinephrine injection. Epinephrine-induced hyperglycemia was mitigated (Fig 3D), and PKA activation was reduced (Fig 3E) in Prom1-/- mice, when compared to that in Prom1+/+ mice. Because immobilization stress causes hyperglycemia via cholinergic muscarinic activation (Tajima, Endo et al., 1996), we performed immobilization test on Prom1+/+ and Prom1-/- mice to test the effect of Prom1 deficiency on the signaling by endogenous epinephrine. We found that the immobilization stress-induced hyperglycemia and PKA activation were decreased in Prom1-/-, when compared to Prom1+/+ mice (Fig 3F and G). However, the reduced responses of Prom1-/- mice to immobilization stress was not due to the different epinephrine levels in the serum of Prom1+/+ and Prom1-/- mice (Fig 3H).
Radixin is the AKAP required for glucagon-induced PKA activation
Because Prom1 deficiency decreased PKA activation in response to glucagon without changing cAMP production (Figs 1 and 2), we postulated that Prom1 might regulate an AKAP which binds to the regulatory subunit of PKA and confines the PKA holoenzyme to a specific cellular location. We identified several AKAPs that are expressed in the liver using an RNA-seq analysis (Fig S3A). Among these AKAPs, AKAP7, 8, 8I, 9, 10, 12 and 13 were selected because they are cytoplasmic or plasma membrane-bound proteins that may interact with Prom1. In addition, Radixin was also selected as the AKAP in question among ERM (ezrin, radixin and moesin) protein family, because radixin is dominantly expressed in hepatocytes (Fig S3B), links actin to the plasma membrane in the liver (Kikuchi, Hata et al., 2002), and is known to function as an AKAP (Gloerich, Ponsioen et al., 2010, Hochbaum, Barila et al., 2011). We knocked down AKAPs expressed in the liver individually and examined their effects on glucagon-induced phosphorylation of PKA substrates in primary hepatocytes to identify which AKAP was regulated by Prom1. Glucagon-induced phosphorylation of PKA substrates was inhibited most markedly by small interfering RNAs (siRNAs) targeting Prom1 or radixin, suggesting that radixin might be an AKAP regulated by Prom1 (Fig 4A and Fig S3C and D). Because ezrin, a well-studied AKAP, was marginally expressed in the liver (Fig S3B), we tested the effect of ezrin knockdown by siRNA on glucagon-induced phosphorylation of PKA substrates as a control. Ezrin deficiency did not change the phosphorylation of PKA substrates (Fig S3E). Glucagon stimulation did not change the amount of radixin, PKA catalytic subunit or regulatory subunit (Fig S3F) as well. Similarly, adenoviral knockdown of radixin or Prom1 using short hairpin (sh) RNA in WT hepatocytes prevented glucagon-induced phosphorylation of CREB and other PKA substrates (Fig 4B, C, Fig S3G and Fig S4A). In contrast, radixin knockdown did not alter glucagon-induced cAMP production (Fig S4B). It has been reported that plasma membrane localization of Epac1, exchange factor directly activated by cAMP, and subsequent Rap1 activation is also mediated by radixin as an AKAP (Hochbaum et al., 2011). We determined Epac1 activity in Prom1-/- primary hepatocytes treated with glucagon. Prom1 deficiency decreased Rap1 activation as well. (Fig S4C). These results suggested that radixin functions as the AKAP regulated by Prom1 in primary mouse hepatocytes.
Next, to determine whether radixin functions as an AKAP during glucagon-induced gluconeogenesis in vivo, we knocked down radixin in mice using adenoviral shRNA, confirmed its transduction efficiency to be close to 100 % in hepatocytes (Fig S4D), and performed the glucagon challenge test. Glucagon-induced hyperglycemia was mitigated by radixin knockdown (Fig 4D). Moreover, radixin knockdown reduced the phosphorylation of CREB and PKA substrates (Fig 4E and Fig S4E), but did not alter cAMP production in the livers of glucagon-treated mice (Fig S4F). Glucose, pyruvate, and insulin tolerance tests were performed on radixin-knockdown mice to further investigate whether radixin regulates hepatic gluconeogenesis in vivo. Radixin knockdown improved glucose and pyruvate tolerance but not insulin tolerance (Fig. 4F), suggesting that radixin knockdown prevented glucagon-induced hepatic gluconeogenesis without affecting insulin signaling.
Prom1 is necessary to confine radixin to the plasma membrane
To determine the mechanism by which Prom1 regulates the AKAP activity of radixin we first examined whether the two proteins were located in close proximity using a proximity ligation assay. Fluorescence signals were observed in Prom1+/+ but not in Prom1-/- primary hepatocytes (Fig 4G, two left panels) and adenoviral overexpression of Prom1 drove the proximity-based fluorescence to reappear in Prom1-/- primary hepatocytes (Fig 4G, two right panels). PKA regulatory subunits were also in close proximity to Prom1 (Fig 4H) implying the formation of Prom1/radixin/PKA complex. Endogenous Prom1 was co-immunoprecipitated with radixin in primary mouse hepatocytes (Fig 4I), suggesting the molecular interaction between Prom1 and radixin. Reciprocal immunoprecipitations of exogenously expressed Prom1 and radixin showed similar results in HEK293 cells (Fig 4J). The molecular interaction between Prom1 and radixin prompted us to speculate that Prom1 is required to confine radixin to the plasma membrane and to link actin to the plasma membrane. We determined the cellular localization of radixin and actin in Prom1+/+ and Prom1-/- primary hepatocytes to test this hypothesis. Radixin and actin were clearly observed in cell-cell contact sites called the canalicular membranes in Prom1+/+ hepatocytes but not in Prom1-/- hepatocytes (Fig 4K). Moreover, the Prom1 deficiency prevented cortical actin from localizing to canalicular membranes, suggesting that Prom1 was required to confine radixin and actin to the plasma membrane. Because Prom1 and ERM proteins are present in detergent-resistant lipid rafts, we speculated that radixin enrichment in detergent-resistant lipid rafts may depend on Prom1 expression. We examined the presence of radixin in detergent-resistant lipid rafts from Prom1+/+ and Prom1-/- primary hepatocytes to test this hypothesis. Prom1-deficient cells exhibited a reduced amount of radixin in the detergent-resistant lipid rafts (Fig 4L). These results demonstrated that Prom1 is required to confine radixin to plasma membrane lipid rafts.
The FERM domain of radixin is necessary for Prom1-dependent gluconeogenesis
To determine which domain in each protein is required for the interaction, we constructed various deletion mutants of both Prom1 and radixin (Fig S5A and B). Co-immunoprecipitation after transient expression of both genes in HEK293 cells showed that the carboxy-terminal tail of Prom1 (IC3) interacted with the FERM domain (1-310) of radixin, because the full-length protein and carboxy-terminal tail of Prom1 co-immunoprecipitated the full-length protein and FERM domain of radixin (Fig 5A and B). Competitive co-immunoprecipitation showed a gradual decrease in the molecular interaction between Prom1 and radixin in proportion to the increasing expression level of the FERM domain in HEK293 cells (Fig 5C), indicating the specific interaction between two proteins through FERM domain. Indeed, the canalicular localization of endogenous radixin disappeared in cells overexpressing the FERM domain (Fig 5D). Next, we monitored glucagon-induced PKA activation after adenoviral overexpression of the FERM domain in primary mouse hepatocytes. Overexpression of the FERM domain prevented glucagon-induced phosphorylation and nuclear localization of CREB (Fig 5E and Fig S5C). Overexpression of the FERM domain prevented glucagon-induced phosphorylation of PKA substrates (Fig 5F), but did not affect glucagon-elicited cAMP production (Fig S5D). We further analyzed the dominant negative effect of the FERM domain on glucagon-elicited gluconeogenesis in vivo. Systemic adenoviral overexpression of the FERM domain mitigated glucagon-elicited hyperglycemia (Fig 5G). In addition, overexpression of the FERM domain prevented the glucagon-induced phosphorylation of CREB and PKA substrates in the livers of glucagon-treated mice (Fig 5H), and improved glucose and pyruvate tolerance but not insulin tolerance (Fig. 5I). Taken together, we concluded that the FERM domain of radixin is necessary for the interaction with Prom1 and its confinement to lipid rafts to facilitate radixin’s function as an AKAP during hepatic gluconeogenesis.
AKAP activity of radixin in glucagon-elicited gluconeogenesis was further substantiated by knockdown-rescue experiment. We examined glucagon-induced PKA activation in primary mouse hepatocytes in which endogenous radixin was knocked down by adenoviral shRNA, and shRNA-resistant radixinR or LPTDR was re-introduced by adenoviral transduction. We used the LPTD mutant of radixin, because L421P mutation disrupts the ability of radixin to bind to PKA, therefore losing AKAP activity and T564D mutation is known to mimic phosphorylation in all ERM proteins in its open and active conformation (Deming, Campbell et al., 2015). Ectopic expression of shRNA-resistant radixinR rescued glucagon-induced phosphorylation of PKA substrates, while radixin mutant LPTDR could not (Fig 6A). Overexpression of LPTDR mutant alone in primary mouse hepatocytes decreased the glucagon-induced phosphorylation of CREB and PKA substrates (Fig 6B), which suggested the dominant negative effect of LPTDR mutant as well.
Because our results showed that Prom1-deficient mice displayed improved glucose and pyruvate tolerance but not insulin tolerance (Fig 2I and J), we examined glucagon-elicited gluconeogenesis in high-fat diet-induced obese (DIO) Prom1+/+ and Prom1-/- mice. DIO Prom1-/- mice exhibited a mitigation of glucagon-elicited hyperglycemia (Fig 6C). The Prom1 deficiency also prevented glucagon-induced phosphorylation of CREB and other PKA substrates (Fig 6D). In DIO Prom1-/- mice, glucose tolerance, but not insulin tolerance, was improved (Fig 6E). Moreover, systemic adenoviral overexpression of Prom1 in DIO Prom1-/- mice enhanced glucagon- and pyruvate-induced hyperglycemia (Fig 6F). These in vivo results showed that the Prom1 deficiency protected mice from diet-induced glucose intolerance
Discussion
Although Prom1 is a cancer stem cell marker located in plasma membrane detergent-resistant lipid rafts, the cellular functions of Prom1 have remained elusive (Krishnan et al., 2013, Roper, Corbeil et al., 2000). For the first time, our study provides new insights into the physiological role of Prom1. We examined the mechanism by which Prom1 regulates hepatic gluconeogenesis using Prom1-deficient mice. The Prom1-radixin axis is a key signaling pathway that regulates cAMP-mediated PKA activation. We propose that Prom1 confines radixin to the plasma membrane and radixin recruits the PKA holoenzyme to the plasma membrane. Subsequently, glucagon-G protein-coupled receptor (GPCR)-Gsα-AC signaling pathway produces cAMP, liberating and activating the PKA catalytic subunit from the PKA regulatory subunit attached to radixin. Because Prom1 deficiency interferes with the function of radixin as an AKAP, Prom1 deficiency prevents cAMP-mediated PKA activation.
ERM family proteins (ezrin, radixin and moesin) act as AKAPs because they interact with the PKA regulatory subunit and various plasma membrane-associated PKA substrates (Neisch & Fehon, 2011). Indeed, ERM proteins are required for PKA-mediated phosphorylation of different membrane-associated proteins, such as β-adrenergic receptor, cystic fibrosis transmembrane conductance regulator (CFTR), Na+-H+ exchanger 3 (NHE3), connexin 43 (Cx43) and exchange protein directly activated by cAMP (EPAC) (Bretscher, Edwards et al., 2002, Fouassier, Duan et al., 2001, Hochbaum et al., 2011, Pidoux, Gerbaud et al., 2014, Sun, Hug et al., 2000, Weinman, Steplock et al., 2003). We focused on the Prom1-radixin complex because among these ERM proteins radixin is the dominant ERM protein in hepatocytes (Kikuchi et al., 2002, Tsukita, Hieda et al., 1989). Radixin knockout mice show hyperbilirubinemia due to loss of multidrug resistance protein 2 (MRP2) from canalicular membranes in the liver (Kikuchi et al., 2002), deafness associated with progressive degeneration of cochlear stereocilia in the inner ear (Kitajiri, Fukumoto et al., 2004), and impaired reversal learning and short-term memory by modulating inhibitory synapse transmission (Loebrich, Bähring et al., 2006). Our study provides the first evidence to show that radixin is also involved in glucagon/β-adrenergic receptor-mediated gluconeogenesis via Prom1-radixin axis. Prom1 deficiency disrupts the localization of radixin in canalicular membranes, prevents the formation of cortical actin (Fig 4K) and inhibits glucagon-, isoprenaline- or cAMP-induced PKA activation (Fig 2, Fig 3, and Fig S2) without changing the amount of PKA catalytic subunit (Fig S3F). These results demonstrate that Prom1 deficiency abrogates the function of radixin as an AKAP which allows the PKA holoenzyme and PKA substrates to assemble in the same place. Failing to confine the PKA holoenzyme and PKA substrates in close proximity due to Prom1 deficiency may explain the reduced phosphorylation level of PKA substrates even at the supra-physiological concentration of activators used in our study. In addition, because PKA has generous substrate specificity (Dalerba et al., 2007), it is crucial to tightly regulate PKA localization through its interaction with AKAPs (Beene & Scott, 2007) and activation through confined cAMP concentration (Rich, Fagan et al., 2001, Zaccolo & Pozzan, 2002). Recently, Smith et al. (Smith, Esseltine et al., 2017) have demonstrated that local PKA activation is regulated by the conformational change of holoenzyme, not by the complete dissociation of catalytic subunit at the physiological concentration of cAMP. In accordance with these reports our findings also demonstrated that local confinement of PKA to which cAMP is readily available by the interaction with radixin, hepatocyte specific AKAP, and Prom1 complex is an important process that provides the specificity in the regulation of glucagon-induced gluconeogenesis. This may explain the lack of action from other AKAP proteins in the presence of glucagon or 8-Br-cAMP. More interestingly, we demonstrated that the importance of Prom1-radixin axis not only in glucagon-elicited gluconeogenesis, but also in β-adrenergic receptor-mediated gluconeogenesis (Fig. 3). These results suggest that Prom1-AKAP interaction may participate in regulating GPCR signaling in other phenotypes or organs.
Membrane-associated AKAPs are known to be involved in cAMP-induced phosphorylation and the activation of nuclear CREB. Although membrane-bound AKAP5 interacts with E-cadherin, β-adrenergic receptor, adenylyl cyclase and the cytoskeleton, cAMP-induced phosphorylation of nuclear CREB is increased by its overexpression but is reduced by its knockdown (Altier, Dubel et al., 2002, Fraser, Cong et al., 2000, Gorski, Gomez et al., 2005). The cAMP-induced phosphorylation of nuclear CREB is also reduced with a cell-permeable peptide treatment that inhibits the molecular interaction between PKA and AKAP (Friedrich, Aramuni et al., 2010, Godbole, Lyga et al., 2017). Similar to the observations in the above examples, the Prom1-radixin complex in the plasma membrane regulates cAMP-induced phosphorylation of nuclear CREB. However, the molecular mechanism by which radixin regulates the phosphorylation of nuclear CREB still remains elusive.
Using Prom1-deficient or radixin-knockdown mice, we show that the Prom1-radixin complex is required for hepatic gluconeogenesis. Because both lean and high-fat diet-fed mice lacking Prom1 or radixin exhibited a decreased blood glucose level after a 4-h fast and improved glucose and pyruvate tolerance without changing insulin sensitivity, Prom1 and radixin may be excellent target proteins for lowering the blood glucose level in patients with diabetes. Therefore, a chemical drug that interferes with the molecular interaction between Prom1 and radixin might thus be useful as a treatment for hyperglycemia by inhibiting gluconeogenic signaling pathway.
Materials and Methods
Animal studies
Prom1 knockout mice were purchased from The Jackson Laboratory (Stock NO. 017743, Bar Harbor, ME, USA). The Prom1-/- mice were backcrossed with C57BL/6N mice for five generations. All animal studies were conducted with the approval of the Korea University Institutional Animal Care and Use Committee and the Korean Animal Protection Law (KUIACUC-2017-14 and -2018-6).
For glucagon stimulation tests, glucagon (200 μg/kg body weight for mice fed a normal chow diet, 100 μg/kg body weight for mice fed a high-fat diet (Sigma-Aldrich, St Louis, MO, USA) was intraperitoneally injected into male mice that had fasted for 4 h. For glucose tolerance tests, D-glucose (2 g/kg body weight) (Sigma-Aldrich, St Louis, MO, USA) was intraperitoneally injected into male mice that had fasted overnight. For insulin tolerance test, insulin (0.75 U/kg body weight for mice fed a normal chow diet, 1.5 U/kg body weight for mice fed a high-fat diet) (Sigma-Aldrich, St Louis, MO, USA) was intraperitoneally injected into male mice that had fasted for 4 h. For pyruvate tolerance test, pyruvate (2 g/kg body weight) (Sigma-Aldrich, St Louis, MO, USA) was intraperitoneally injected into male mice that had fasted overnight. For epinephrine stimulation test, epinephrine (3µg/10g) (Sigma-Aldrich, St Louis, MO, USA) was intraperitoneally injected into male mice that had fasted for 4h. For immobilization stress, male mice were restrained in a ventilated acrylic restrainer fit to allow the animal to breathe but not to move otherwise.
Preparation of mouse primary hepatocytes
Primary hepatocytes were isolated from 8-week-old C57BL/6 male mice as previously described (Koo, Flechner et al., 2005). Briefly, mice were anesthetized with avertin (intraperitoneal injection of 250 mg/kg body weight), and livers were perfused with a pre-perfusion buffer (140 mM NaCl, 6 mM KCl, 10 mM HEPES, and 0.08 mg/mL EGTA, pH 7.4) at a rate of 7 mL/min for 5 min, followed by a continuous perfusion with collagenase-containing buffer (66.7 mM NaCl, 6.7 mM KCl, 5 mM HEPES, 0.48 mM CaCl2, and 3 g/mL collagenase type IV, pH 7.4) for 8 min. Viable hepatocytes were harvested and purified with a Percoll cushion. Then, hepatocytes were re-suspended in complete growth medium, 199 medium containing 10% FBS, 23 mM HEPES and 10 nM dexamethasone, and seeded on collagen-coated plates at a density of 300,000 cells/mL. After a 4-h attachment period, the medium was replaced with complete growth medium before use in any experiments and changed daily.
Measurement of cAMP concentrations
Levels of cAMP were quantified using a cAMP ELISA kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s protocol. For mouse primary hepatocytes, cells growing in 6-well plates were lysed with IBMX, and cAMP concentrations were quantified. Concentrations of cAMP in mouse liver samples were measured using an ELISA and normalized to the wet liver weight.
Measurement of PKA activity
PKA activity was measured using a PKA kinase activity assay kit (Abcam, Cambridge, UK), according to the manufacturer’s protocol. Briefly, whole-cell lysates were incubated with a specific synthetic peptide as a substrate for PKA and a polyclonal antibody that recognizes the phosphorylated form of the substrate. Relative PKA activity was measured by determining the optical density.
Proximity ligation assay
An in situ proximity ligation assay was used to detect protein-protein interactions in cells. Briefly, cells were cultured on collagen-coated confocal dishes and fixed with 4% paraformaldehyde. Then, cells were permeabilized with 0.1% Triton X-100 in phosphate buffer and incubated with blocking buffer for 30 min to prevent nonspecific binding. Samples were sequentially incubated with a primary antibody, PLA probe, ligase, and polymerase, according to the manufacturer’s instructions. PLA-positive cells exhibited red fluorescent signals. The fluorescent signal was observed using a confocal laser scanning microscope (Carl Zeiss 700, 40 X water objectives).
Quantitative real-time PCR
RNA (2 μg) was reverse transcribed to cDNAs using random hexamer primers, oligo dT and Reverse Transcription Master Premix (ELPIS Biotech, Daejeon, Korea). Quantitative real-time PCR analyses were performed using the cDNAs from the reverse transcription reactions and gene-specific oligonucleotides (Appendix Table S2) in the presence of TOPreal qPCR 2X premix (Enzynomics, Daejeon, Korea). The following PCR conditions were used: an initial denaturation step at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 58°C for 15 s and elongation at 72°C for 20 s. The melting curve for each PCR product was assessed for quality control. Supplementary Table 2 shows the sequences of the primers used for qPCR.
Measurement of hepatic glucose output
Glucose production was determined using the glucose assay kit from Sigma-Aldrich according to the manufacturer’s instructions. Primary hepatocytes were seeded on collagen-coated 6-well plates (0.8 x 106 cells per well). After 3 h, cells were infected with a virus overnight. After 24 h, the medium containing the virus was removed and replaced with fresh 199 medium. After 18 h, the medium was removed, and the cells were rinsed twice with PBS. Then, glucose production buffer (consisting of glucose-free DMEM lacking phenol red, pH 7.4, 20 mM sodium pyruvate, 2 mM l-glutamine and 15 mM HEPES) was added to the cells. After 4 h, the medium was collected and the amount of glucose in the medium was determined using the glucose assay kit (Sigma-Aldrich, St Louis, MO, USA).
siRNA interference
All siRNAs (Appendix Table S3) were synthesized by Bioneer Inc. (Daejeon, Korea). Cells were transfected with 100 nM siRNA using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, USA).
Adenovirus preparation and infection
Adenoviruses harboring sh-Con, sh-Rdx (radixin), sh-Prom1, GFP and FERM were produced as previously described (Yi, Park et al., 2013). AD293 cells were re-infected with viral stocks to amplify the viruses, and viruses were purified by double cesium chloride-gradient ultracentrifugation. Infectious viral particles in the cesium chloride gradient (density = ∼1.345) were collected, dialyzed against a 10 mM Tris (pH 8.0), 2 mM MgCl2 and 5% sucrose solution and stored at -80°C. Recombinant adenovirus (0.5 × 109 pfu) was injected into the tail veins of mice. Four days after the injection, the mice were subjected to blood glucose metabolism tests.
Isolation of detergent-resistant lipid rafts
Livers from wild type and Prom1 knockout mice were lysed in 1 mL of lysis buffer (1% Brij 35, 25 mM HEPES, pH 6.5, 150 mM NaCl, protease and protease inhibitor cocktail) and subjected to discontinuous sucrose gradient ultracentrifugation (40%, 30%, and 5%) using a SW41 Ti rotor (39,000 rpm) for 18 h at 4°C. After centrifugation, the sucrose solutions were fractionated into 12 fractions. An opaque buoyant band corresponding to the lipid rafts was collected at the interface between the 30% and 5% sucrose solutions.
Plasmid construction and transient transfection
Deletion mutants of Prom1-Flag and Myc-radixin (Appendix Table S4) were generated by reverse PCR using the primer sets (Appendix Table S5). mTagBFP2-Farnesyl-5 was a gift from Michael Davidson (Addgene plasmid # 55295). The Myr-BFP-IC3 construct of Prom1 was generated as follows: first, the myristoylation sequence was added at the N-terminus of pmTagBFP2 using reverse PCR; second, Prom1-tail-3xFlag was added at the C-terminus of pmTagBFP2 using the DNA assembly method. Myr-BFP was generated by removing the Prom1-tail from Myr-BFP-IC3 by reverse PCR. DNA transfection was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, USA), according to the manufacturer’s protocol.
Immunoblotting and immunofluorescence staining
Cells were lysed with the following lysis buffer: 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor mixture and phosphatase inhibitor mixture (Sigma-Aldrich, St Louis, MO, USA). Whole cell lysates obtained from the supernatant after microcentrifugation at 14,000 rpm for 15 min at 4°C were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a nitrocellulose membrane and incubated with specific primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. Antigens were visualized using an enhanced chemiluminescence substrate kit (Thermo Fisher Scientific, Waltham, MA, USA). For immunoprecipitation, cells were lysed in buffer containing 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 and a protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). Whole-cell lysates (500 μg of protein) were incubated with specific antibodies overnight and then with 60 μL of a slurry of Protein A- or Protein G-agarose beads (Roche, Mannheim, Germany) for 3 h. Immunoprecipitates were analyzed by immunoblotting. For immunofluorescence staining, cells were fixed with PFA and stained with antibodies against the indicated protein.
Immunohistochemistry
Each paraformaldehyde-fixed samples were either embedded in paraffin or frozen in OCT compound and cut into 5um-thick sections. Tissue samples were then stained with hematoxylin-eosin (HE) or periodic acid-schiff (PAS) according to standard protocol, and the images were captured on light microscope (Leica). For Prom1 immunohistochemistry, fresh-frozen tissues post-fixed in 4% paraformaldehyde. Tissue sections were immunostained with antibody directed against prominin-1 (Thermo Fisher Scientific, Waltham, MA, USA). The samples were pretreated with 2.5% horse serum for 20min to block nonspecific antibody binding and incubated with the antibodies of interest for overnight at 4°C. The slides were then treated with Rhodamine-conjugated secondary antibody. After mounting with fluorescence mounting medium (Agilent, Santa Clara, CA, USA), the samples were captured on an LSM 800 META confocal microscope (Zeiss, Oberkochen, DE).
Rap activation assay
Rap activity assays were performed as manufacture’s protocol (Abcam; ab212011, Cambridge, UK). Briefly, primary mouse hepatocytes were lysed at 4℃ in a buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor mixture and phosphatase inhibitor mixture. Cells lysates were incubated for 30 min with agarose beads coupled to the Rap-binding domain (RBD) of RalGDS (Ral Guanine Nucleotide Dissociation Stimulator), which bind specifically to the active form of Rap. Subsequently, the precipitated GTP-Rap was detected by western blot analysis using anti-Rap1 antibody.
Statistical analysis
Values are presented as means ± SEM. A two-tailed Student’s t-test was used to calculate the P values
Acknowledgement
We thank all members of our laboratory for their supports and intellectual inputs during the preparation of this manuscript. Funding: This work was supported by grants from the National Research Foundation of Korea awarded to; Y.-G. Ko (2015R1A5A1009024), H. Lee (2017R1A6A3A01009334) and S. Lee (2017R1D1A1A02017979), and partially by grant from Korea University to H. Lee. Author contributions: Study concept and design, HL, DY, SL and YK; acquisition of data, HL, DY, JP and HYL; analysis and interpretation of data, HL, DY, JL, SL and YK; material support, SK and KS; drafting of the manuscript, SL and YK. YK is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Conflicts of interest: The authors disclose no conflict of interests.
Abbreviations
- AKAP
- A kinase-anchored protein
- AC
- adenylyl cyclase
- cAMP
- cyclic adenosine monophosphate
- PKA
- protein kinase A
- CREB
- cAMP response element binding protein
- pck
- phosphoenol pyruvate carboxykinase
- g6p
- glucose-6-phosphatase
- G6pc
- g6p catalytic subunit
- PI3K
- phosphatidylinositide 3-kinases
- Fbp1
- fructose 1,6-bisphosphatase 1
- GPCR
- G protein– coupled receptor
- PDE
- phosphodiesterase
- IBMX
- 3-isobutyl-1-methylxanthine
- 8-Br-cAMP
- 8-Bromo-cAMP
- DIO
- diet-induced obese
- shRNA
- short hairpin RNA
- FERM
- F for 4.1 protein, E for ezrin, R for radixin and M for moesin
- CFTR
- fibrosis transmembrane conductance regulator
- NHE3
- Na+-H+ exchanger 3
- Cx43
- connexin 43
- EPAC
- exchange protein directrly activated by cAMP