CREB3 protein family: the promising therapeutic targets for cardiovascular and metabolic diseases

Abstract

Significant advancements in cardiovascular and metabolic disease research have been made with the CREB3 protein family. Studies have revealed that members of this family are crucial in the development of these diseases, contributing to the regulation of lipid metabolism, inflammation, and vascular function. These studies provide useful information for future therapeutic strategies in cardiovascular and metabolic diseases.

Graphical abstract

Introduction

Cardiovascular and metabolic diseases are primarily characterized by a relatively clear cause-and-effect relationship between cardiovascular damage and metabolic abnormalities. It excludes etiologies such as structural cardiovascular disease, arrhythmias, and cardiomyopathy, with atherosclerotic cardiovascular disease (ASCVD) as the primary manifestation, with coronary heart disease, sudden cardiac death, and heart failure as the primary outcomes. It is a clinical syndrome in which intervention in metabolic disorders can effectively improve the prognosis, and has also been referred to as cardiovascular metabolic syndrome. (Govindarajan et al. 2005) Metabolic factors such as obesity, diabetes mellitus, and dyslipidemia are often clustered together, these factors are not only cardiovascular risk factors but also involved in the pathophysiological process of target organ damage, which is the causative factor leading to the occurrence and development of ASCVD. (Alberti et al. 2009) This shows that metabolism is becoming increasingly important for the development of cardiovascular disease.

Metabolism is a general term for a series of chemical reactions in living organisms, including the processes of energy production and utilization, synthesis, and decomposition of substances, which are necessary for the maintenance of life. When metabolic balance is disrupted and homeostasis is compromised, signaling pathways that aim to restore equilibrium are believed to respond to stress conditions, such as endoplasmic reticulum and Golgi stress. The CREB3 protein family, as a transcription factor anchored to the endoplasmic reticulum, plays a critical role in regulating cardiovascular and metabolic diseases.

The CREB3 protein family is involved in processes such as cholesterol synthesis, (Nakagawa et al. 2016; Ruppert et al. 2019) lipid metabolism, (Kang et al. 2017; Khan & Margulies 2019; J. H. Lee et al. 2011) and glucose regulation. (H. Kim, Zheng, Walker, Kapatos, & Zhang, 2017; M. W. Lee et al. 2010) These proteins regulate the expression of genes essential for maintaining normal cardiovascular functions, including cholesterol homeostasis and glucose regulation. In addition, the CREB3 protein family is involved in the process of neovascularization and repair in the cardiovascular system, (Liu, Liu, Liu, Niu, & Liu, 2023) and regulates the inflammatory response to cardiovascular disease. (Luebke-Wheeler et al. 2008; K. Zhang et al. 2006) Therefore, this review will focus on the cardiovascular implications of the regulation of the CREB3 protein family in metabolism. We expect that the CREB3 protein family will provide rational therapeutic options for cardiovascular and metabolic diseases sometime in the future.

A brief introduction to the CREB3 protein family

CREB3 is a class of transcription factors characterized by the basic leucine zipper domain, which has a bearing on various biological processes such as cell stress, ER stress, Golgi stress, energy metabolism, and differentiation. (Gao et al. 2021; Sampieri et al. 2019; Smith et al. 2022) The CREB3 protein family, widely expressed in humans, consists of DNA-binding proteins. This family includes CREB3 along with its subtypes—CREB3L1, CREB3L2, CREB3L3, and CREB3L4—each exhibiting distinct transcriptional activity. (Yuxiong et al. 2023) Each member of the CREB3 protein family is localized within lumen segments and follows a consistent sequence of regulated intramembrane proteolysis (RIP), mediated by Site-1 protease (S1P) and Site-2 protease (S2P). This process facilitates protein cleavage in specific transmembrane regions, leading to their activation. The sequence, from the N-terminal (cytoplasmic side) to the C-terminal (medial cavity), includes: A transcriptional activation domain that enhances DNA binding in a sequence-specific manner; A unique region (approximately 30 residues) at the N-terminal end of the basic leucine zipper (bZIP) domain, possibly conferring a specialized function to these factors; The bZIP domain itself, consisting of basic and leucine zipper motifs, which recognizes specific DNA sequences and promotes dimerization; The transmembrane domain (TMD), anchoring the protein to the endoplasmic reticulum (ER) membrane. (Miotto & Struhl 2006) The CREB3 protein family shares significant sequence similarity with ATF6, as both contain a transmembrane domain that allows binding to the endoplasmic reticulum (ER). Additionally, both families possess a transcriptional activation domain that recognizes specific DNA sequences and a basic leucine zipper (bZIP) domain, which facilitates dimerization. But unlike ATF6, ER localization of CREB3 is determined by the cytoplasmic domain of the ER retention motif (ERM), which is highly conserved in all CREB3 members but absent in ATF6. (Bailey et al. 2007) Under normal conditions, the full-length form of the ER-anchored uncut transcription factor remains inactive. The rapid and seasonable response to ER stress can be guaranteed by RIP activation; Therefore, RIP is the rate-limiting step in transcription factor activation. (E, Zhang, Zhou, & Wang, 2014) During ER stress, CREB3 is transported to the Golgi apparatus, where it is sequentially cleaved by S1P and S2P. This cleavage releases N-terminal fragments containing transcriptional activation and bZIP domains. These released fragments then move to the nucleus, where they activate the transcription of target genes. CREB3 can recognize various sequences, including the cAMP response element (CRE), the ER stress response element II (ERSE-II), and the unfolded protein response element (UPRE). (Raggo et al. 2002; Taniguchi & Yoshida 2017) CREB3 transcription factors bind to target DNA sites as either homodimers or heterodimers, thereby influencing the transcriptional regulation of various genes. (Miotto & Struhl 2006) Although these proteins are structurally similar to each other, they differ markedly in their activation stimuli, tissue distribution, and response element binding (Fig. 1).

Fig. 1.

A Gene structure of the CREB3 protein family (B). CREB3 acts as an endoplasmic reticulum anchoring protein, is transported to the Golgi and is sheared by S1P and S2P and delivers the N-terminus to the nucleus and functions as a transcription factor

CREB3 in Cardiovascular and metabolic diseases

CREB3 is ubiquitous and has been detected in all human adult and fetal tissues examined. (Lu et al. 1997) The CREB3 protein is responsive to a variety of stimuli, including stress within the endoplasmic reticulum and Golgi apparatus, and is activated by the protein RIP. (E et al. 2014).

Obesity

The unfolded protein response (UPR), activated by endoplasmic reticulum stress, is essential for managing glucose and lipid homeostasis. CREB3, a key transcription factor in the unfolded protein response (UPR), when present at reduced levels, leads to heightened energy expenditure and a higher respiratory exchange rate. This reduction in CREB3 expression also provides protection against weight gain from a high-fat diet (HFD), mitigates basal hyperglycemia, and reduces sex-specific lipid accumulation in tissues. This indicates that CREB3 plays a pivotal role in managing diet-induced obesity and energy metabolism. Its involvement underscores the need for further research to explore its potential as a therapeutic target for addressing metabolic disorders. Nevertheless, CREB3 does not significantly affect the plasma lipid profile. (Smith et al. 2022).

Hepatic steatosis

In addition to its role in endoplasmic reticulum stress, CREB3 is also crucial in managing Golgi stress. In response to Golgi stress, the CREB3 pathway triggers apoptosis by enhancing the transcription of ADP-ribosylation factor 4 (ARF4), a key small GTPase involved in Golgi cargo transport. When Golgi dispersing compounds induce stress, CREB3 is mobilized from the endoplasmic reticulum to the Golgi. There, it undergoes cleavage by S1P and S2P before translocating to the nucleus. This process upregulates ARF4 expression, causing partial fragmentation of the Golgi and eventually leading to apoptosis. Although Golgi stress is known to activate CREB3, the exact mechanism behind this phenomenon is still unclear. (Gao et al. 2021) When cells are under Golgi stress, CREB3 is broken down into smaller pieces and modified by a chemical process called phosphorylation, which stops it from being destroyed by the cell's waste disposal system. The stabilized N-terminal fragment of CREB3 is subsequently translocated to the nucleus, where it directly interacts with the ApoA-IV promoter. This leads to the activation of the ApoA-IV gene, which increases the uptake of free fatty acids from the peripheral tissues, promoting hepatic steatosis. (Kang et al. 2017) Lipotoxicity and glucotoxicity induce the expression of CREB3 and ARF4, which are important mediators of Golgi stress response leading to apoptosis. However, simple glucotoxicity does not increase the transcription of CREB3 or ARF4 in cellular models. This suggests that CREB3 is more sensitive to lipotoxicity. The effects of increased transcript levels of CREB3 and ARF4 caused by lipotoxicity and glucose toxicity on the organism's phenotype are still unknown. Lipotoxicity and glucose toxicity cause Golgi stress and activate CREB3 according to the Golgi stress program. CREB3, in turn, upregulates ARF4, which causes apoptosis. However, it is currently unclear whether other factors regulate ARF4 (Fig. 2). (Başçıl Tütüncü et al. 2022).

Fig. 2.

A CREB3 is ubiquitous and has been detected in all human adult and fetal tissues examined. B Golgi stress causes activation of CREB3. C Activated CREB3 binds to target genes and exerts corresponding biological effects: CREB3 binds to the ApoA-IV gene and promotes the expression of ApoA-IV, leading to an increase in peripheral free fatty acid uptake; CREB3 binds to the ARF4 gene and promotes the transcription of ARF4, leading to apoptosis; and CREB3 binds to the C/EBP components of CCR2, ultimately leading to atherosclerosis. D TNF-α activates the NF-κB pathway and promotes the expression of the CREB3 variant sLZIP, leading to increased expression of MMP-9, increased migration of VSMCs, and ultimately atherosclerosis

Atherosclerosis

CREB3 controls the expression of chemokine receptors CCR2 and CCR1, which are involved in the initial phases of atherogenesis in a human monocyte cell line (THP-1). It influences MCP-1-driven monocyte chemotaxis and Lkn-1-mediated chemotaxis, both of which are critical for atherosclerosis development. As a transcription factor, CREB3 interacts with the C/EBP element of CCR2, enhancing its transcriptional activation and thereby playing a crucial role in the progression of atherosclerosis. (Sung et al. 2008).

sLZIP, a variant of CREB3, functions primarily as a transcription factor. During fasting, cyclic adenosine monophosphate inhibits sLZIP degradation resulting in increased expression of phosphoenolpyruvate carboxylase and glucose-6-phosphatase in hepatocytes. This promotes glucose production and indicates that sLZIP regulates gluconeogenic enzyme expression. These findings indicate that sLZIP is vital for preserving glucose homeostasis during periods of starvation. (Kang et al. 2021) The cytokine TNF-α, which is known for its pro-inflammatory effects, can activate NF-κB signaling and increase the expression of sLZIP. This, in turn, triggers the transcription of macrophage matrix metalloproteinase-9 (MMP-9) and enhances its protein hydrolysis activity, leading to increased migration of vascular smooth muscle cells (VSMCs) and eventually contributing to the development of atherosclerosis. Given its crucial role in atherosclerosis, sLZIP could be a promising target for therapeutic interventions aimed at treating this disease. (J. Kim & Ko 2014).

Cardiac remodeling

Upregulation of CREB3 is closely associated with the formation of endothelial cells. When overexpressed in endothelial progenitor cells (EPCs), CREB3 enhances the expression of the HO-1 protein, which is essential for both angiogenesis and cell survival, and AKT phosphorylation. This, in turn, reduces myocardial swelling and dysfunction in spontaneously hypertensive (SHR) rats, thereby attenuating cardiac hypertrophy (Fig. 3). (Liu et al. 2023).

Fig. 3.

Decreased CREB3 expression increases energy expenditure and respiratory exchange rate and protects mice from HFD-induced weight gain, basal hyperglycemia, and sex-specific tissue lipid accumulation. In MEF cells, knockdown of CREB3 resulted in increased mitochondrial ATP production, increased mitochondrial and endoplasmic reticulum Ca2 + efflux, and elevated reactive oxygen species levels. Overexpression of CREB3 in EPCs led to increased HO-1 protein expression, promotion of angiogenesis, improved cell survival as well as AKT phosphorylation, and alleviation of cardiac hypertrophy and dysfunction caused by spontaneous hypertension

Oxidative stress

Recent research has revealed that silencing CREB3 in MEF cells results in elevated basal levels of reactive oxygen species (ROS) and greater sensitivity to H₂O₂. Additionally, this knockdown increased mitochondrial ATP production and cellular respiration, along with a higher efflux of ER-Ca²⁺ and mitochondrial Ca²⁺. These findings suggest that CREB3 may have an inhibitory metabolic potential during times of cellular stress, thus protecting MEF cells from oxidative stress. (Smith et al. 2023).

Diabetes

CREBRF is a cellular protein that negatively regulates CREB3, promoting its protein degradation by recruiting it out of the nucleus. (Audas et al. 2008)Obesity and diabetes are conditions that are strongly affected by genetics, but the specific genes that contribute to these conditions are not yet well understood. In humans, a variant in the Creb3 regulatory factor (CREBRF) gene—known as rs373863828 (p.Arg457Gln); CREBRFR457Q—has been linked to increased odds of obesity but reduced odds of diabetes. Although the mechanism of action of CREBRF is not yet known, recent research suggests that it may contribute to the body's adaptation to nutritional stress by regulating gene expression downstream of TORC1. (Kanshana et al. 2021).

In conclusion, the targeted knockdown of CREB3 has proven to be an effective strategy for mitigating the negative impacts of metabolic disorders, including obesity, hepatic steatosis and diabetes mellitus. In the context of cardiovascular disease, CREB3 has been observed to promote the expression of chemokine receptors CCR1, CCR2, and MMP-9, which may contribute to the progression of atherosclerosis. Additionally, in the context of myocardial remodeling and impaired cardiac function resulting from spontaneous hypertension and myocardial ischemia/reperfusion injury, which are influenced by ROS, CREB3 may exert a beneficial effect. In addition, CREB3 may play a beneficial role in the remodeling and impaired cardiac function associated with spontaneous hypertension, as well as in myocardial ischemia/reperfusion injury, which is significantly influenced by ROS.

CREB3L1 /CREB3L2 in Cardiovascular and metabolic diseases

The gene named CREB3L1 exhibits high expression levels within numerous tissues, including the pancreas, prostate, and bone. (Murakami et al. 2009; Omori et al. 2002) Recent research on mouse models indicates that CREB3L1 is crucial for bone formation, with osteoblasts showing high levels of CREB3L1 expression and an expanded rough endoplasmic reticulum that accumulates significant amounts of bone matrix proteins. However, the underlying cause of this phenotype in osteoblasts related to CREB3L1 remains unclear. It may be attributed to the inactivation of the target genes of CREB3L1 that are essential for the transportation of proteins from the bone matrix from the endoplasmic reticulum to the Golgi apparatus. However, the direct targeting of the type I collagen gene Col1a1 by CREB3L1 has been discovered. Its trans-activation occurs via an enhancer element located in the osteoblast-specific promoter of Col1a1. Therefore, CREB3L1 plays a crucial role in bone formation, likely by stimulating the transcription of Col1a1 and facilitating the secretion of bone matrix proteins. (Murakami et al. 2009) CREB3L1 is expressed in pancreatic β-cell lines and rodent islets, exhibiting elevated activity during pancreatic development. Introducing the active form of CREB3L1 into pancreatic β-cell lines resulted in the enhanced expression of genes related to protein transport, such as Kdelr3, COPζ2, and Sv2c, as well as genes involved in extracellular mesenchymal production, including Papss2, Matn1, and Chst12. These observations suggest that CREB3L1 proteins may play a significant part in pancreas development. (Vellanki et al. 2010).

CREB3L2 has widespread expression, with the highest levels in the placenta, lung, spleen, intestine, and cartilage. (Saito et al. 2009; Storlazzi et al. 2003) A research paper studying mice with knocked out Creb3l2 gene concluded that CREB3L2 is vital for the formation of cartilage. The study demonstrated that Sec23a, a component of the protein complex II (COPII) that facilitates the transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus, is affected by CREB3L2. (Saito et al. 2009) Furthermore, considering its notably elevated expression in subcutaneous adipose tissue, it is necessary to conduct further research on the plausible involvement of CREB3L2 in lipid metabolism. (Khan & Margulies 2019).

It has been noted that CREB3L1 and CREB3L2 play important roles in the secretory pathway of non-secretory cells. (Fox et al. 2010) Despite the lack of studies of CREB3L1 and CREB3L2 in cardiovascular and metabolic diseases, linking cardiovascular disease to the secretory pathway in the future is not out of the question, given the important roles that both play in the secretory pathway.

CREB3L3 in Cardiovascular and metabolic diseases

CREB3L3 has a liver-specific expression. (Chin et al. 2005; Omori et al. 2001; K. Zhang et al. 2006) However, CREB3L3 is also expressed in the stomach and small intestine. (J. H. Lee et al. 2011).

In metabolomics analyses, research has shown that CREB3L3 targets or potentially targets genes that are involved in various functions related to cardiovascular metabolism. These functions encompass lipoprotein metabolism (Apoc2, Apoa5, Apoa1, Scarb1), fatty acid binding (Fabp2), lipid storage (Cidec), desaturation and elongation of fatty acids (Fads1, Fads2, Elovl2, Elovl5), gluconeogenesis (Pck1, G6pc), and fatty acid oxidation (Cpt1a). (Ruppert et al. 2019).

Endoplasmic reticulum stress induces the activation of estrogen-related receptor-γ (ERRγ), an orphan nuclear receptor. ERRγ interacts with the transcriptional coactivator PGC1α, which subsequently attaches to the ERRγ response element (ERRE) on the CREB3L3 promoter. This process causes histone acetylation at the ERRE of the CREB3L3 promoter, leading to the upregulation of CREB3L3 expression. (Misra et al. 2014) After translation, the full-length CREB3L3 undergoes post-translational modification through N-linked glycosylation, which regulates RIP cleavage and activates CREB3L3. The modified protein then binds to target genes to perform its biological functions. (Bailey et al. 2007; Chan et al. 2010) It has been found that certain dietary factors, such as an atherogenic high-fat (AHF) diet, (H. Kim et al. 2014a, b; C. Zhang et al. 2012) Western diet,(Xu et al. 2014) and long-term treatment of HFD (Xu et al. 2014; Xu et al. 2015) can lead to the expression and activation of CREB3L3. Additionally, CREB3L3 activation was also observed during periods of starvation in fasting/refeeding experiments. During fasting, CREB3L3 was found to be acetylated and during feeding, it was deacetylated. These processes took place in a time-dependent manner and were mediated by the lysine acetyltransferase P300/CREB-binding protein-associated factor as well as the histone deacetylase sirtuin-1(SIRT1), respectively. (H. Kim et al. 2015a, b) However, it has also been shown that endoplasmic reticulum pressure is dispensable for CREB3L3 activation in the liver (Fig. 4). (Xu et al. 2014).

Fig. 4.

The way CREB3L3 is activated. ERRγ binds to PGCIα and acts on the ERRE of the CREB3L3 gene to promote CREB3L3 activation. Both fasting and HFD treatment can lead to CREB3L3 activation. Refeeding inhibits CREB3L3 activation

Atherosclerosis

ApoA is a protein produced by the liver and intestines, primarily responsible for transporting lipid molecules into the bloodstream. In Hep-G2 cells cultured in the liver, CREB3L3 expression enhances the synthesis and secretion of specific carriers, including ApoA-I and ApoA-IV. (Barbosa et al. 2013) ApoA-IV activates lecithin-cholesterol acyltransferase to transfer cholesterol to HDL particles, regulating HDL metabolism. (Chen & Albers 1985; Steinmetz & Utermann 1985) Research conducted on mice has found that overexpression of ApoA-IV can prevent the development of atherosclerosis. (Cohen et al. 1997; Duverger et al. 1996; Ostos et al. 2001) ApoA-IV levels were elevated in hepatic steatosis or steatohepatitis, but not fully correlated with CREB3L3(N). (Xu et al. 2014) Overexpression of CREB3 has been demonstrated to activate the transcription of ApoA-IV as well. (Kang et al. 2017; Sanecka et al. 2012) This may be due to the physical and functional interaction between CREB3 and CREB3L3, resulting in synergistic regulation of ApoA-IV gene expression. Further experiments are required to confirm this hypothesis. ApoC-II and ApoA-V are synthesized in hepatocytes and are involved in promoting TG hydrolysis in VLDL particles by stimulating peripheral lipoprotein lipase (LPL) activity. CREB3L3 enhances LPL activity, which in turn increases TG clearance by up-regulating the hepatic expression of apolipoproteins ApoA-IV, ApoA-V, and ApoC-II (Fig. 5). (Wade et al. 2021).

Fig. 5.

CREB3L3 ameliorates atherosclerosis by promoting apolipoprotein expression

Studies have shown that CREB3L3 has an impact on triglyceride metabolism in the liver. In mice lacking the Creb3l3 gene, the levels of circulating triglycerides were found to be higher compared to wild-type mice. Furthermore, microarray analysis revealed that besides the genes responsible for apolipoproteins, other genes involved in triglyceride metabolism such as Fgf21, a known regulator of plasma triglycerides, were also affected. (J. H. Lee et al. 2011) The synergistic activation of hepatic fibroblast growth factor 21 (FGF21) expression by CREB3L3 and peroxisome proliferator-activated receptor alpha (PPARα) has been demonstrated. (H. Kim et al. 2014a, b; Nakagawa et al. 2014) The synthesized FGF21 protein is released into the bloodstream and transported to peripheral tissues, where it exerts thermogenic physiological effects. (Fisher et al. 2012)and inhibits lipolysis. (Park et al. 2016a, b) FGF21 inhibits the development of atherosclerosis by decreasing vascular smooth muscle cell proliferation, hypercholesterolemia, and oxidative stress, through lipocalin-dependent and lipocalin-independent mechanisms. (Kokkinos et al. 2017; Lin et al. 2015) Recent research indicates that the metabolic phenotypes seen in LDLR-/-CREB3L3-/- mice are linked to lower plasma levels of FGF21. However, the lack of FGF 21 in LDLR-/-Tg CREB3L 3 mice resulted in the amelioration of atherosclerosis, which dispels the hypothesis that FGF 21 is a major contributor to anti-atherosclerosis. (Nakagawa et al. 2021) N-glycosylation increases the transcriptional activation potential of CREB3L3, affecting the production of PPARα, a key regulator of hepatic mitochondrial fatty acid β-oxidation, and the activity of stearoyl coenzyme A desaturase-1 (SCD-1), a significant hepatic lipogenesis enzyme via modulating their transcriptional activity and protein interactions. These effects ultimately lead to reduced lipid deposition and attenuation of lipotoxicity. (N. Zhang et al. 2020) PPARα and CREB3L3 play distinct roles in liver lipid metabolism, with their effects largely influenced by dietary conditions, despite both contributing to the activation of the FGF21 pathway. Studies indicate that the absence of CREB3L3 impacts PPARα signaling in two key ways during a ketogenic diet: First, it lowers the expression of PPARα and its associated genes involved in fatty acid oxidation and ketogenesis. Second, it significantly activates the hepatoproliferative function of PPARα. (Ruppert et al. 2019)This reinforces the possibility that CREB3L3 may have a major role in atherosclerosis. (Park et al. 2016a, b) The SEL1L-HRD1 complex, a highly conserved component of the mammalian endoplasmic reticulum-associated degradation system, is essential for regulating FGF21 transcription. It achieves this by controlling the ubiquitination and degradation of the ER-resident transcription factor CREB3L3, thereby influencing its nuclear levels. It is worth noting that the SEL1L-HRD1 ERAD complex does not affect PPARα, another well-known FGF21 transcription factor. These findings indicate that there is a physiologically regulated negative correlation between SEL1L-HRD1 ERAD and CREB3L3-FGF21 levels, particularly under fasting and growth conditions (Fig. 6). (Bhattacharya et al. 2018).

Fig. 6.

Activated CREB3L3 regulates common pathways of lipid metabolism and glucose metabolism

Sterol regulatory element binding proteins (SREBP) are transcription factors that maintain lipid homeostasis by regulating the expression of enzymes involved in the synthesis of cholesterol, fatty acids, triacylglycerols, and phospholipids. When sterol levels drop, the precursor is cleaved, activating cholesterol-producing genes to restore balance, similar to the activation mechanism of CREB3. SREBP-1c mainly governs fatty acid synthesis and insulin-induced glucose metabolism, especially in adipogenesis, while SREBP-2 is primarily involved in cholesterol production. SREBP-1a isoforms, however, seem to participate in both pathways. The regulation of SREBP-1c isoforms occurs primarily through insulin at the transcriptional level, while the regulation of SREBP-1a and SREBP-2 is mainly sterol-dependent. (Eberlé et al. 2004) Although CREB3L3 and SREBP share similar modes of activation, they are in functional competition with each other. Specifically, CREB3L3 causes the retention of SREBP-1c in the endoplasmic reticulum by upregulating the SREBP-insulin-induced gene-1 complex and its sister protein, Insig-2a. Additionally, CREB3L3 competes with SREBP in the Golgi for the cleavage of S1P and S2P. Thus, a deficiency in CREB3L3 may lead to the activation of genes related to SPREBP, which is accompanied by a decrease in TG catabolism and an increase in lipogenesis. This results in a considerable accumulation of triglyceride-rich remnant lipoproteins and severe hypertriglyceridemia. Furthermore, the absence of intestinal CREB3L3 has been observed to increase cholesterol absorption and SREBP-2 activation in the liver, resulting in a significant enrichment of cholesterol in these lipoproteins, which is a critical factor in the development of atherosclerosis. (Nakagawa et al. 2021; Wang et al. 2016).

A recent paper found that mice overexpressing CREB3L3 specifically in the small intestine (Creb3l3 Tg mice) and fed a lithogenic diet (LD) had lower plasma cholesterol and hepatic lipid levels compared to wild-type mice. In addition, these mice experienced a reduction in the formation of gallbladder cholesterol crystals, while displaying an increase in fecal cholesterol output. The study's findings propose that augmented expression of Creb3l3 in the intestine can lessen cholesterol absorption, hence lowering blood cholesterol levels. The expression of Npc1l1, the protein responsible for cholesterol transportation in the intestine, was demonstrated to be reduced in the small intestine of Creb3l3 Tg mice. These mice also showed reduced levels of other proteins engaged in cholesterol transport, namely Abca1, Abcg5/8, and Srb1. The study demonstrated that the regulation of Npc1l1 expression is directly modulated by CREB3L3. Conversely, mice devoid of CREB3L3 exhibited enhanced intestinal Npc1l1 expression, resulting in increased levels of cholesterol in both the liver and blood, whilst exhibiting decreased cholesterol excretion in the feces. (Kikuchi et al. 2016) This aligns with findings following hepatic Creb3l3-specific knockdown in cholesterol metabolism. However, regulation differs as hepatic CREB3L3 regulates FGF21 to inhibit Srebf2 expression (encoding the SREBP2 protein) in the liver. This ultimately reduces cholesterol synthesis and maintains cholesterol homeostasis; (Nakagawa et al. 2016) In contrast, intestinal CREB3L3 balances cholesterol by regulating the rate-limiting transporter protein NPC1L1, which mediates intestinal cholesterol absorption. This explains why liver-specific knockout mice had lower plasma FGF21 levels than FLOXED mice, while intestinal-specific knockout mice did not. (Nakagawa et al. 2016) In addition, knocking down Creb3l3 in the liver increased plasma cholesterol but not liver cholesterol, whereas knocking it down in the intestine elevated both plasma and liver cholesterol. (Kikuchi et al. 2016; Nakagawa et al. 2016).

The preceding analysis allows us to draw the following conclusions: Activation of CREB3L3 has been demonstrated to exert a significant inhibitory effect on the development of atherosclerosis. This is achieved by promoting the production and secretion of apolipoproteins, which facilitate the clearance of triglycerides (TG). Furthermore, CREB3L3 has been shown to interact with PPARα to stimulate FGF21 expression, thereby inhibiting the progression of atherosclerosis. Additionally, CREB3L3 has been observed to compete with sterol regulatory element-binding protein (SREBP) for the S1P and S2P binding sites, promoting lipid metabolism and alleviating the advancement of atherosclerosis. Nevertheless, although the upregulation of ApoA4 expression by CREB3L3 can facilitate peripheral fatty acid uptake and alleviate hyperlipidemia, excessive accumulation of ApoA4 will also inevitably result in fatty lesions in the liver (Fig. 7).

Fig. 7.

Consequences of the lack of CREB3L3, including liver-specific knockout and small intestine-specific knockout

Lipid metabolism/glucose metabolism

Fat-specific protein 27 (Fsp27) has been observed to associate with lipid droplets, and it is thought that this interaction plays a crucial role in supporting their growth and triglyceride storage in white adipocytes. Additionally, Fsp27 is highly expressed in steatotic livers, contributing to triglyceride accumulation. CREB3L3 regulates the protein, and fasting reduces hepatic Fsp27 in mice lacking Creb3l3. Nevertheless, Fsp27 deletion does not result in reduced fasting liver triglyceride levels. CREB3L3 regulates triglyceride homeostasis through various mechanisms, and it is unclear how decreasing Fsp27 expression affects triglyceride accumulation in Creb3l3-deficient mice. Inhibition of fatty acid oxidation and triglyceride secretion, caused by CREB3L3 loss, leads to triglyceride build-up in the liver, which may be more significant than the effects of impaired Fsp27 expression. (Xu et al. 2015) All of the findings indicate that CREB3L3 plays a significant function in lipid metabolism, which cannot be disregarded.

CREB3L3 plays a crucial role in the regulation of normal physiological processes that require elevated secretory function, including gluconeogenesis and the secretion of products specific to the liver and intestine. In regard to the fasting response, elevated levels of nuclear CREB3L3 collaborate with PPARα (Ruppert et al. 2019; Vecchi et al. 2014) to promote the expression of gluconeogenic genes, such as PEPCK-C and G6Pase. Additionally, in diabetic mice receiving Creb3l3 RNAi treatment, there was a significant reduction in fasting blood glucose levels. (M. W. Lee et al. 2010) The activation of CREB3L3 by fasting and/or circadian rhythms additionally stimulates glycogenolysis via increased Pygl expression. Concurrently, upregulation of Pck1 and G6pc by CREB3L3 promotes gluconeogenesis in the same conditions. (H. Kim et al. 2017a, b) These findings suggest that CREB3L3 plays a significant role in activating the gluconeogenic program in fasted mice. Further evidence suggests that glucose metabolism is regulated in a circadian manner by CREB3L3 through the BMAL1-AKT-glycogen synthase 3β (GSK3β) cascade. (Zheng et al. 2016) Lysine acetylation is a crucial process for the transcriptional activity of CREB3L3, which displays a typical circadian pattern. It reaches its maximum level from 10:00 to 14:00 and exhibits resilience at 2:00 a.m. Furthermore, the dynamic expression pattern of Pck1 and G6pc mRNAs corresponds to the circadian rhythm of CREB3L3 and demonstrates its role in regulating glucose metabolism. (H. Kim et al. 2017a, b) It has been posited that ferredoxin functions as a gluconeogenesis sensor in starved mice, and starved Creb3l3 knockout mice exhibit markedly reduced blood glucose levels relative to starved wild-type mice, (Vecchi et al. 2014) indicating that when the expression of iron-modulating hormones is reduced, the body is unable to detect gluconeogenic signals, which consequently results in lower blood glucose levels. However, research has demonstrated that CREB3L3 triggers the activation of genes associated with gluconeogenesis. Conversely, when CREB3L3 is knocked out, this leads to the down-regulation of gluconeogenic genes, thereby reducing blood glucose levels. (M. W. Lee et al. 2010) So it remains to be demonstrated whether iron modulators play a direct role in glucose metabolism.

CREB3L3 then triggers the activation of SAP and CRP genes, which are key players in the acute phase response, along with other genes that potentially impact lipid metabolism. During inflammation, the liver increases the production of acute phase response (APR) proteins like serum amyloid P (SAP) and C-reactive protein (CRP). This effect is primarily controlled by the cleavage and activation of CREB3L3 by inflammatory cytokines, which then drive the transcription of both SAP and CRP. Additionally, the coexpression of CREB3L3 and ATF6 significantly boosts APR gene expression, suggesting that the formation of a heterodimer between these factors enhances the transcriptional activity of APR genes. (Luebke-Wheeler et al. 2008; K. Zhang et al. 2006) Among these biomarkers, C-reactive protein serves as a robust indicator of cardiovascular events. Additionally, it is noteworthy that this biomarker can be used for multiple purposes. (Ridker et al. 2002) One of these is driving the development of atherosclerosis, including arterial thrombosis (Devaraj et al. 2009) and endothelial dysfunction. (Hein et al. 2009) Hepcidin is a liver-derived peptide essential for maintaining iron balance and is a key acute-phase response gene, upregulated by proinflammatory cytokines. CREB3L3 is involved in regulating iron metabolism and frequently forms heterodimers with other bZip factors. Notably, CREB3L3 interacts with XBP-1 to co-activate the hepcidin promoter, enhancing its expression. (Vecchi et al. 2009).

In addition to its crucial function in lipid metabolism, CREB3L3 plays a pivotal role in regulating glucose metabolism in a circadian pattern, as well as activating gluconeogenesis during fasting conditions. Furthermore, CREB3L3 may also stimulate the production of acute-phase proteins, thereby facilitating the development of atherosclerosis.

Nonalcoholic steatohepatitis

In a nonalcoholic steatohepatitis (NASH) model, CREB3L3 promotes SIRT3 expression which is a crucial regulator of protein deacetylation in mitochondria. This process controls MnSOD deacetylation and restrains the inflammatory vesicle activation of NOD-like receptor Pyrin domain 3 (Nlrp3). Consequently, this safeguards the hepatocytes from oxidative stress. (J. Zhang et al. 2022).

Previous studies have mainly concentrated on the function of CREB3L3's N-terminal shear fragment. However, there has been little research on the carboxy-terminal fragment of CREB3L3, known as CREB3L3-C, derived from membrane-bound full-length CREB3L3. Recent studies have shown that CREB3L3-C is secreted as a hepatic factor in response to fasting or hepatic stress. Efficient secretion of CREB3L3-C via cytokinesis requires CaMKII-mediated phosphorylation of CREB3L3-C. The formation of the ANGPTL3-ANGPTL8 complex inhibits LPL activity in mice. Secreted CREB3L3-C blocks the formation of this complex, which leads to increased LPL activity in plasma and metabolic tissues. This results in the attenuation of hypertriglyceridemia and hepatic steatosis. (H. Kim, Song, Zhang, Davies, & Zhang, 2023).

Considering that lipotoxicity and non-resolved oxidative stress are important contributors to metabolic inflammation, and since CREB3L3 is unexpectedly responsible for protecting both lipid metabolism and oxidative stress, the future of CREB3L3 research in cardiovascular and metabolic diseases looks promising.

It is proposed that CREB3L3-N may protect hepatocytes from oxidative stress by promoting SIRT3 expression. Furthermore, it is suggested that CREB3L3-C may enhance LPL activity and alleviate hypertriglyceridemia and hepatic steatosis by inhibiting the formation of the ANGPTL3-ANGPTL8 complex (Fig. 8).

Fig. 8.

A Stimulated by inflammatory cytokines, CREB3L3 promotes the progression of atherosclerosis. B CREB3L3 plays a protective role in NASH disease models. C Lipid-lowering effects exerted by CREB3L3-C

CREB3L4 in cardiovascular and metabolic diseases

The function of CREB3L4 seems to be associated with its tissue expression. Research shows that this transcription factor is involved in the proliferation of prostate cancer cells, a process facilitated by androgen receptor (AR) and IRE1α. (T. H. Kim, Park, Kim, & Ahn 2017a, b) Additionally, CREB3L4 fosters the advancement of breast cancer in humans and impacts the production of sperm in mice. (Adham et al. 2005; Pu et al. 2020).

Myocardial ischemia–reperfusion injury

Our team previously discovered that the hexosamine biosynthetic pathway (HBP) is responsible for producing uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which helps modify O-linked GlcNAc (O-GlcNAc) proteins. This modification increases the survival rate of cells when they encounter lethal stress. Recent studies have revealed that CREB3L4 can bind directly to the conserved UPRE of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) promoter. This, in turn, enhances the HBP flux and O-GlcNAc protein modification. Tisp40 is a transcription factor that is commonly found in cardiomyocytes and is associated with the UPR. Targeting Tisp40 could lead to the development of effective methods for reducing cardiac I/R injury. (X. Zhang et al. 2023).

Obesity/diabetes

CREB3L4 is involved in adipocyte differentiation. Its absence induces differentiation by stabilizing CCAAT/enhancer-binding protein (C/EBPα) or reducing the expression of GATA3, a negative regulator of PPARγ2 expression. This consequently leads to an increase in the expression of peroxisome proliferator-activated receptor γ (PPARγ2) and C/EBPα. However, observations of smaller adipocytes in high-fat diet (HFD)–fed mice without CREB3L4 are made, although no significant effects are evident on overall body weight or fat mass. Nevertheless, adipocyte proliferation results in enhanced glucose tolerance and insulin sensitivity. (T. H. Kim et al. 2014a, b; Lundgren et al. 2007; McLaughlin et al. 2007) Various dietary conditions and age may have diverse impacts on CREB3L4 function. Studies indicate that mice with Creb3l4 KO, fed with a low-fat diet (LFD), and those who are aged, present augmented hypertrophic white adipose tissue (WAT), thereby causing lowered insulin sensitivity, impaired glucose tolerance, and bigger adipocytes. However, it remains ambiguous whether CREB3L4 bolsters adipocyte differentiation. (T. H. Kim et al. 2015a, b) Studies have demonstrated that hypertrophic adipocytes, namely, larger fat cells, may exhibit distinct biochemical features that differ from those of smaller adipocytes. Such differences may involve elevated lipolysis, heightened production of inflammatory cytokines, and reduced secretion of anti-inflammatory adipokines such as leptin and lipocalin. These variations could cause inflammation in adipose tissue due to greater mechanical and hypoxic stress on hypertrophic adipocytes. (Ghaben & Scherer 2019) Adipocyte hypertrophy results in an increase in circulating free fatty acids, which can be observed in individuals who have a high risk of cardiovascular disease (Fig. 9). (Rydén & Arner 2017).

Fig. 9.

A Mechanisms of CREB3L4 protection against myocardial ischemia–reperfusion injury. B Creb3l4 knockout mice under different conditions

Discussion

In summary, CREB3 may have a potential role in the uptake of free fatty acids, apoptosis, and the progression of atherosclerosis. Therefore, we can speculate that reducing the expression of CREB3 could ameliorate cardiac diseases caused by lipid metabolism disorders. Although CREB3 has been shown to promote ApoA-IV expression which is responsible for the uptake of free fatty acids in the peripheral tissues, the effect of this process on peripheral lipid accumulation and the subsequent development of atherosclerosis is still unknown. Further research is necessary to investigate this phenomenon. Given the continued maturation of gene editing technology and the systemic distribution of CREB3, we should further explore whether cardiomyocyte-specific knockdown of CREB3 would have a more dramatic effect than that obtained by systemic knockdown. In patients with spontaneous hypertensive heart disease and ischemia/reperfusion injuries who do not have risk factors for lipid disorders, CREB3 may play a positive role, but it may cause some damage to the liver and increase the likelihood of fatty liver disease. However, in patients with both lipid metabolism disorders, spontaneous hypertension, and ischemia–reperfusion, it is important to identify the primary causative factors before considering how to modify CREB3 expression for treatment.

In the above discussion, we can clearly see that CREB3L3 plays a beneficial role in lipid metabolism disorders, and activated CREB3L3 promotes lipid metabolism, reduces peripheral lipid accumulation, and is effective in delaying the progression of atherosclerosis; however, as in the case of CREB3, the continuous accumulation of ApoA-IV leads to an increased load on the liver. In addition, we found an interesting phenomenon: under the same fasting condition, the CREB3L3 glycogenolysis and gluconeogenesis pathways were significantly activated, whereas the lipid metabolism pathway was inhibited; therefore, fasting does not seem to be an optimal choice for those who rely on dietary improvements to regulate lipid metabolism. In addition to this, CREB3L3 can alleviate oxidative stress. However, since CREB3L3 has not yet been studied in cardiomyocytes, further research is still needed to determine whether it will be protective in patients with myocardial ischemia–reperfusion injury. Under the stimulation of inflammatory cytokines, CREB3L3 and ATF6 nevertheless exerted a role in promoting atherosclerosis, which contradicts the previous conclusions. This may be due to temporal differences, with atherosclerosis promotion dominating during the acute response, gradually changing to atherosclerosis amelioration over time. Nevertheless, further investigation is required to substantiate this hypothesis (Fig. 10).

Fig. 10.

Summary of the role of the CREB3 protein family in cardiovascular and metabolic diseases

Conclusion

The CREB3 protein family has been investigated in connection with cardiovascular metabolic ailments and was discovered to be of great importance in modulating numerous physiological pathways. These pathways incorporate cholesterol biosynthesis, lipid metabolism, and glucose regulation, underscoring the multiplicity of functions that this family has in preserving the regular performance of the cardiovascular system. Moreover, the role of the CREB3 protein family extends to regulating intracellular calcium balance, contributing to vascular neovascularization and repair mechanisms, as well as playing a part in myocardial protection and inflammatory response. These functions demonstrate the intricate and interconnected network of regulation orchestrated by the CREB3 protein family.

The research indicates that the CREB3 protein family is implicated in various stages of cardiovascular and metabolic disease development, providing insights for better comprehension of these ailments. Therefore, comprehending the molecular mechanisms of these proteins is crucial for the advancement of more efficient therapeutic approaches.

Author contributions

Yi-Peng Gao and Xin Zhang designed this study and guided the entire process. Yi-Peng Gao and Can Hu wrote the manuscript. Yi-Peng Gao and Min Hu prepared Figures 1-10. Yu-Xin Hu and Wen-Sheng Dong edited the original draft. Yun-Jia Ye and Kang Li revised the manuscript and participated in the preparation of the review. All authors made intellectual contributions and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the Natural Science Foundation of Hubei Province (No. 2023AFB099), the Fundamental Research Funds for the Central Universities (No. 2042023kf0046), the Open Project of Hubei Key Laboratory (No. 2023KFZZ028) and Clinical Medicine + Youth Talent Support Program of Wuhan University.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

Not applicable.

Competing interests

The authors declare no competing interests.