Research progress on key genes of vitamin D signaling pathway

LIU Kun, LIU Jun-lin

1.Department of Dermatology, The Second Affiliated Hospital of Hainan Medical University, Haikou 570200, China

2.Department of Dermatology and Venereology, Hainan Provincial People's Hospital, Haikou 570200, China

Keywords:

ABSTRACT In recent years, more and more reports have been reported on the extracellular role of vitamin D signaling pathway.Studies have shown that both genomic and non-genomic effects of vitamin D signaling pathway are involved in calcium homeostasis, skeletal effects, cell proliferation, differentiation, oxidative stress, angiogenesis, inflammatory response, immune regulation, production and recognition of antimicrobial peptides to varying degrees.This paper reviews the research progress of key genes such as GC, CYP2R1, CYP24A1, CYP27B1, VDR and RXRα in vitamin D receptor signaling pathway, in order to understand the physiological and pathological mechanisms of this pathway more accurately and comprehensively, and lay a foundation for further research.

1.Introduction

In recent years, research on the outside the bone effects of vitamin D has continued to deepen, and the understanding of vitamin D and its signaling pathways has also deepened.This article reviews relevant research both domestically and internationally in recent years and summarizes the roles of some key genes in the vitamin D receptor (VDR) signaling pathway in vitamin D synthesis and metabolism, in order to lay a foundation for further research.

2.Biological functions of vitamin D and its active forms

Vitamin D3(VD3), generated by 7-dehydrocholesterol (7-DHC)in the skin, is a natural form of vitamin D present in the human body.Under the action of ultraviolet rays, 7-DHC generates vitamin D3precursor, which is then isomerized into vitamin D3[1].The synthesis ability of vitamin D3in the skin is directly proportional to the intensity of ultraviolet rays[2].In addition to the skin synthesis pathway, vitamin D3can also be converted from some natural foods or chemical compounds.Vitamin D3itself does not possess biological activity, and its main active form in the body is 1,25(OH)2D3[3].1,25(OH)2D3is a product of vitamin D3through biochemical reactions in the body.The intermediate metabolite 25(OH)D3is commonly used to evaluate the level of vitamin D in the body[4].

Vitamin D3and its derivatives can regulate the growth,development, metabolism, and others of the human body.They can also regulate about 5% of genes in the entire genome through genomic effects, and can directly affect the function of over 900 genes[5].

3.Key genes in the vitamin D signaling pathway

At present,GC, CYP2R1, CYP24A1, CYP27B1, VDR, andRXRα genes are the majority of research on vitamin D metabolism and vitamin D signaling pathways.This article will review the research progress of the key genes mentioned above.

3.1 CYP2R1 gene

TheCYP2R1gene is located on chromosome 11p15.2,approximately 21 kb long and contains 9 exons, encoding the 25-hydroxylase of vitamin D.The CYP2R1 gene is mainly expressed in the liver and its main function is to hydroxylate vitamin D3at the 25-C position, converting vitamin D3into 25(OH)D3.There are also studies indicating that theCYP2R1gene can be expressed in human testes.When its expression is impaired, it can lead to a significant decrease in serum 25(OH)D3and a decrease in bone density[6].Hector F.DeLuca’s team from the University of Wisconsin confirmed that the serum levels of 25(OH)D3in CYP2R1 gene knockout mice were significantly reduced, but the synthesis of 25(OH)D3was not completely blocked, indicating the presence of other genes encoding 25-hydroxylase[7].

The Tom D Thacher team[8,9] sequenced the promoter, exon,and intron exon flanking regions of theCYP2R1gene from 12 Nigerian families, confirming that theCYP2R1mistranslated mutations (L99P and K242N) were associated with vitamin D levels.Immunofluorescence technology was used to label the protein content of their transcripts, confirming that these two mutations can significantly reduce the 25(OH)D3content in vivo, And it cannot be improved by supplementing vitamin D (P=0.025 for homozygous and heterozygous mutations, and P=0.008 for the control group).The atypical vitamin D deficiency rickets caused by this mutation belong to the subtype 1B of vitamin D dependent rickets.A genomewide association analysis of a large sample of 33 996 European Americans showed a significant correlation between rs10741657 of theCYP2R1gene and human vitamin D deficiency (P=3.3×10-20)[10].Professor Min Gao’s team in China conducted[11] a multi-stage cluster sampling survey on more than 1800 Uyghur and Kazakh residents in Xinjiang.Using logistic regression analysis, it was found that the single nucleotide polymorphism (SNP) rs10766197 (G/A)of the CYP2R1 gene was associated with vitamin D deficiency in the Uyghur population (OR=6.533, P=0.019).A 2-year multicenter clinical study from the United States involving[12] 732 obese patients from white, black, Latino, and other ethnicities found that CYP2R1 rs10741657 (GG/GA/AA) had similar gene frequencies between males and females, high protein and low protein diet groups, and high fat and low fat diet groups, while there were differences among different ethnicities (P0.05), And there is a correlation between different genotypes and fasting blood glucose levels (P=0.004).The Spanish Xavier Matias Guiu team[13] used immunohistochemical methods to study the paraffin embedded samples of 217 endometrial cancer patients retained at the University of Arno in Barcelona,Leleda Hospital and São Paulo Hospital.It was found that vitamin D can partially increase the production of 25(OH)D3in the tumor through CYP27A1 and CYP2R1, thereby enhancing the effect of VDR through autocrine/paracrine methods to delay the progression of endometrial cancer and enhance its anticancer effect.

3.2 GC gene

TheGCgene is located on chromosome 4q13.3, approximately 64 kb long, and contains 15 exons, encoding the vitamin D binding protein (VDBP).The main physiological function of VDBP is to bind with vitamin D and its metabolites in serum and transport them to different target organs to produce corresponding effects.For example, transporting vitamin D to the liver to synthesize 25(OH)D3, and transporting 25(OH)D3to the kidneys to further synthesize active derivatives.

There are three common variants of VDBP, Gc1f, Gc1s, and Gc2.The different variants are determined by two SNPs on the 11th exon of the GC gene: rs7041 (Gc1f and Gc1s) and rs4588 (Gc1f and Gc2)[14].The differences in amino acid sequences of different variants of VDBP result in their different affinity with vitamin D, with Gc1f having the highest affinity for vitamin D metabolites and Gc2 having the lowest[15].

Genome wide association studies have shown[16] that rs7041 and rs4588 are associated with free 25(OH)D3levels; Individuals carrying the rs7041 (Gc1s) TT genotype and the rs4588 (Gc2) AA genotype have lower serum levels of 25(OH)D3[17].These SNPs have varying proportions among different ethnic groups, resulting in black and yellow people often expressing Gc1f type VDBP, while white people are more likely to express Gc1s type VDBP, and Gc2 type is more common among yellow people and white people in Europe,and is rare among black people[14,18].In vitro studies have shown[19]that in monocytes, high affinity VDBP(Gc1f)reduces the effect of 25(OH)D3on gene expression compared to low affinity VDBP(Gc1s or Gc2).This means that the VDBP variant may affect the bioavailability of 25(OH)D3.Multiple studies have suggested[20-22]that rs7041, rs4588, rs2282679, rs1155563, rs12512631, rs17467825,rs2298850, and rs3755967 of the GC gene are all associated with 25(OH)D3levels.A prospective study in Denmark involving 1743 participants showed[23] that the GC genotype of rs4588 may affect the risk of colorectal cancer by affecting the intake of vitamin D in the digestive system (IRR=0.91, 95%CI: 0.82-1.01).A large-scale cross-sectional study from Brazil showed that the CC genotype of rs2282679 and the Gc2 subtype of the GC gene were associated with lower VDBP and total 25(OH)D3levels, as well as higher risk of vitamin D3deficiency (P<0.001 and 0.01, respectively).

In addition, studies have found that activated T cells have VDBP expression on their surface, which can regulate the expression of downstream genes in the vitamin D pathway, and the balance between regulatory T cells and inflammatory T cells is also affected by VDBP[24].Some scholars have found through the study of pneumonia mouse models that VDBP can bind fatty acids and play an important role as a chemokine in neutrophil aggregation[25].

3.3 CYP27B1 gene

TheCYP27B1gene is located on chromosome 12q14.1,approximately 4.7 kb long and contains 9 exons, encoding vitamin D 1 α- Hydroxylase.This enzyme is mainly present in the kidneys and its function is to convert 25(OH)D3into 1,25(OH)2D3which results in biological effects.

Research based on patients with rickets has found that inactivation or deletion of the CYP27B1 gene can cause type I vitamin D dependent rickets (VDDR1), also known as pseudo vitamin D deficiency rickets[26].This result was confirmed by another similar study: mice withCYP27B1gene knockout exhibited rickets like symptoms and were unable to detect 1,25(OH)2D3, as well as secondary hypocalcemia and hyperparathyroidism[27,28].In addition to being expressed in the kidneys, theCYP27B1gene can also be expressed in the skin and parathyroid glands, and is negatively regulated by its product 1,25(OH)2D3[29].There are also reports[30] that the expression of CYP27B1 gene has been found in macrophages or cancer cells of normal individuals and individuals with autoimmune diseases (such as Crohn’s disease, sarcoidosis,etc.).Unlike in the kidneys and parathyroid glands, the expression products of CYP27B1 gene in macrophages and cancer cells are not regulated by serum 1,25(OH)D3levels, but are upregulated by immune stimuli (such as IFN-γ, LPS, IL-15,etc.).A study by Boston University Medical Center in Massachusetts, USA, showed that CYP27B1 can convert 25(OH)D3in human adipocytes into 1,25(OH)2D3, thereby inhibiting the production of inflammatory cytokines and reducing the risk of obesity related metabolic diseases by inhibiting fat burning[31].There have been studies monitoring the expression of this gene in the placenta of pregnant women, and the authors think it may be related to immune function[32].However,under normal physiological conditions, the functional effects of its expression outside the kidneys still need further research.

A recent report[33] suggests that CYP27B1 gene is associated with colorectal cancer, prostate cancer, breast cancer, lung cancer,pancreatic cancer, non Hodgkin’s lymphoma, oral lichen planus and multiple sclerosis.Among them, rs10877012 (promoter C>A)is associated with the risk of primary chronic adrenal insufficiency(OR=1.53, 95% CI 1.07-2.19, P=0.02).In another study, it was found[34] that rs118204009 may be associated with the occurrence of multiple sclerosis.

3.4 CYP24A1 gene

The CYP24A1 gene is located on chromosome 20q13.2,approximately 28kb long and contains 13 exons, encoding vitamin D 24 hydroxylase.This enzyme is a rate limiting enzyme for vitamin D synthesis and belongs to the cytochrome P450 family.It is mainly present in the liver, kidneys, and all cells containing VDR.Its main function is to hydroxylate 25(OH)D3into 24,25(OH)2D3and 1,25(OH)2D3[35], which has functions such as maintaining the normal operation of the vitamin D endocrine system and regulating the body’s estrogen and its receptors[36].

Animal experiments have shown that[37]: 50% of mice with CYP24A1 gene deletion die before 3 weeks of age, while surviving mice cannot clear 1,25(OH)2D3in their bodies, which can cause intramembrane bone damage.However, when both CYP24A1 and VDR genes are deleted together, the bone damage in mice is actually reduced.This suggests that the increase in 1,25(OH)2D3functioned by VDR is the cause of bone defects.A study from Canada on four families with idiopathic infantile hypercalcemia showed[38] that the inactivation mutation ofCYP24A1may be one of the causes of idiopathic infantile hypercalcemia.Sequence analysis showed that the CYP24A1 gene in these children produced five different mutations [E143del (in box deletion of E143), E322K, R396W,L409S, and R159Q].All mutations can affect the function of proteins.All mutated expression products had their catabolic ability disrupted, with only L409S mutation maintaining a small amount of activity.Other studies have shown[39] that theCYP24A1mutation also affects calcium metabolism in adults.These patients are characterized by hypercalcemia, hypercalciuria, and recurrent kidney stones.Therefore, when patients consider long-term hypercalcemia associated with kidney stones, attention should be paid to the possibility ofCYP24A1mutations, especially when supplementing with vitamin D or similar drugs[40].

Domestic scholars have used SELDI to study the polymorphism of theCYP24A1gene in healthy Chinese Han population.The results indicate that[41] rs1570669, rs34043203, rs3787557, and rs6068816 have a higher distribution frequency in healthy Chinese Han population, and the GCTT haplotype frequency is the highest.In recent years, some studies have shown[42] thatCYP24A1gene polymorphism is associated with the occurrence and development of tumors, and is significantly associated with tumor dedifferentiation and poor prognosis.A study on female lung cancer found that the rs6068816 T allele can reduce the risk of lung adenocarcinoma and other types of advanced lung cancer in non-smoking women(OR=0.59, 95%CI=0.35-0.99,P=0.048).In addition, the rs6068816 T allele is also a protective factor in the presence of oil fume exposure.Non smoking women with the CC genotype have a 2.32 fold higher risk of lung cancer compared to individuals with the CT and TT genotypes (95% CI: 1.31-4.10, P=0.004).And this study concluded through Meta-analysis that the rs6068816 TT genotype is associated with a lower overall risk of cancer, while the rs4809957 AG genotype is associated with a higher overall risk of cancer.At the same time, CYP24A1 gene also plays an important role in the development and prognosis of breast cancer through vitamin D signaling pathway.In the study of 1102 breast cancer patients in the TCGA database[43]: low CYP24A1 gene expression is significantly related to poor prognosis of breast cancer (P<0.0001).Recent studies have shown[44] that individuals with the CYP24A1 gene’s rs1570669 AG genotype have a 28% lower risk of developing urinary system tumors compared to the GG genotype (OR=0.72,P=0.016)[44].In the dominant model, individuals with the AGAA genotype have a significantly lower risk of developing urinary system tumors compared to the GG genotype (OR=0.74, 95%CI=0.57-0.94, P=0.015).In addition, studies have also found that theCYP24A1gene is associated with the occurrence of colorectal cancer.CYP24A1is a highly sensitive intestinal epithelium specific marker, indicating that detecting the expression of CYP24A1 can help differentiate colon tumors[45].The above research suggests that further exploration is needed for the related progress ofCYP24A1gene polymorphism in China.

3.5 VDR gene and Retinoid X Receptor α (RXRα) gene

In the related research of vitamin D signaling pathway,VDRandRXRα gene has been a lot of genetic research, and the relationship between the two is also relatively close.The VDR gene is located on chromosome 12q13.11, approximately 63kb long, and contains 12 exons.The coding product VDR belongs to the steroid receptor family and is widely distributed in organs, tissues, and nucleated cells throughout the body.RXRα gene is located on chromosome 9q34.2, approximately 114 kb long, and contains 12 exons.Its coding product the RXRα, It can function by combining with VDR to form heterodimers.

The SNP research on VDR genes is the most common, and VDR gene polymorphisms vary to varying degrees among different races,ethnicities, and regions.Different SNPs ofVDRgenes can affect vitamin D transport and metabolic pathways, and are associated with susceptibility to many diseases[46].In multiple mouse experiments usingVDRgene knockout and/or vitamin D deficiency, it has been shown[47-49] that vitamin D and VDR genes play important regulatory roles in maintaining gastrointestinal epithelial and barrier functions.

RXRα is ligand dependent transcription factors in the nuclear receptor superfamily, composed of the A/B region (regulating the N-terminal domain or AF-1 domain), DNA binding domain (DBD),hinge region, and ligand binding domain (LBD)[50].Scholars have confirmed the characteristics of VDR/RXR complexes using small angle X-ray scattering and hydrogen deuterium exchange technology, and confirmed the synergistic effect between VDR/DBD DNA and VDR/LBD DNA, indicating that ligands and DNA can jointly affect the expression of microgenes[51,52].And RXR is also a ligand activated transcription factor that plays a role in cell differentiation, growth, and apoptosis.Two studies have explored the relationship between the SNPs ofRXRα gene and vitamin D levels in healthy individuals.The Matthias Wjst team believes that there is a significant correlation between rs3132299 and 1,25(OH)2D3levels,as well as rs877954 and 25(OH)D3levels[53].However, Jiyoung Ahn et al.showed that polymorphism in RXRα is not associated with levels of 25(OH)D3or 1,25(OH)2D3[54].

4.Conclusion and Prospect

The vitamin D signaling pathway extensively regulates gene expression and is also a key pathway for the physiological effects of vitamin D and its derivatives.At the genetic level, this pathway is involved in regulating about 5% of the entire genome, directly affecting the function of over 900 genes[5].It plays an important role in biological processes such as calcium homeostasis, skeletal effects,cell proliferation, differentiation, oxidative stress, angiogenesis,inflammatory response, immune regulation, production and recognition of antimicrobial peptides[5,55,56].This article reviews the research progress of six key genes in this pathway, with the aim of gaining a more accurate and comprehensive understanding of the physiological and pathological mechanisms of this pathway, laying the foundation for further research.