Alkaline phosphatase (ALP; EC 3.1.3.1) is a zinc-containing enzyme that is widely distributed across the biological world, from bacteria to higher animals. It has an optimal pH in the alkaline range and catalyzes the hydrolysis of phosphate esters into inorganic phosphate and alcohol. The basic structure of ALP is conserved from Escherichia coli to humans, suggesting that it evolved from a common ancestor. Through the hydrolysis of phosphate esters, ALP plays a role in various physiological processes in living organisms.

In humans, ALP is classified into at least four isoforms: tissue-nonspecific ALP (TNSALP), which is expressed in bone, liver, and kidney; intestinal-type ALP (IAP), which localizes to the small intestine; placental ALP; and germ cell ALP [1], [2], [3], [4]. All isoforms are considered to share common physiological functions because they participate in phosphate metabolism by hydrolyzing phosphorylated compounds and providing inorganic phosphate.

Human TNSALP is encoded by the liver/bone/kidney ALP (ALPL) gene (Gene ID: 249) located on chromosome 1, which consists of 12 exons. TNSALP in bone tissue is known as a “bone formation marker”, and studies on hypophosphatasia, a disorder caused by loss-of-function mutations in the ALPL gene, demonstrated that TNSALP plays a crucial role in the mineralization of bone tissue [5], [6].

In contrast, IAP highly localizes to the brush border membrane of intestinal epithelial cells and is recognized as an “intestinal differentiation marker” [7], [8]. Human IAP is encoded by the Intestinal Alkaline Phosphatase (ALPI) gene (Gene ID: 248) located on chromosome 2, which consists of 11 exons (Fig. 1). Furthermore, human IAP shares 86.5 % amino acid sequence homology with human placental ALP and 56.6 % with TNSALP [9].

One of the physiological functions of IAP is its role as a mucosal defense factor through the dephosphorylation of lipopolysaccharide (LPS), a bacterial endotoxin, which contributes to the regulation of intestinal inflammation [10]. In addition, IAP has been shown to ameliorate colitis in mice via protective effects mediated through the Toll-like receptor 4 pathway, which serves as a receptor for LPS [11].

Knockout mice lacking intestinal ALP were recently generated and achieved greater weight gain and higher serum triglyceride levels when fed a high-fat diet than wild-type mice, indicating the critical role of IAP in lipid metabolism [12]. One proposed mechanism is that IAP promotes the uptake of long-chain fatty acids in the intestines by dephosphorylating cluster of differentiation 36 (CD36), a scavenger receptor involved in long-chain fatty acid transport [13]. Furthermore, the oral administration of IAP to mice has been shown to attenuate metabolic syndrome [14].

In animal experiments, we previously demonstrated that the intake of dietary factors, such as lipids, lactose, and fat-soluble vitamins, increased intestinal ALP activity, showing a strong relationship between IAP, dietary components, and lipid metabolism [15], [16], [17], [18]. Previously, we investigated the relationship between fat-soluble vitamins and IAP using Caco-2 cells [19], [20].

Furthermore, using Caco-2 cells, a human colon carcinoma-derived cell line, we investigated the relationship between fat-soluble vitamins and IAP [19], [20]. Caco-2 cells differentiate into intestinal epithelium-like cells when cultured and are widely used as a cell model to screen intestinal functions, including the inhibitory effects of compounds on glucose absorption and the intestinal membrane permeability of drugs [21]. Caco-2 cells grown in vitro under standard culture conditions in the absence of inducers of differentiation spontaneously exhibits enterocyte-like differentiation and polarization [22]. As human primary enterocytes cannot be obtained in large numbers, Caco-2 cell monolayers are widely utilized for drug absorption studies as a model of the human small intestine. In Caco-2 cells, differentiation is characterized by high activity levels of brush border-associated enzymes such as ALP [22]. We showed that vitamin K (menaquinone-4) and an active form of vitamin D, 1-alpha, 25-dihydroxyvitamin D3 [1, 25(OH)2D3], both fat-soluble vitamins, enhanced ALP activity and ALPI gene expression in Caco-2 cells. These findings indicate that these nutritional factors are closely associated with the maintenance of intestinal homeostasis [19], [20].

As described above, increasing attention has been paid to the multifunctional physiological roles of IAP, not only in phosphate metabolism, but also in maintaining intestinal homeostasis, lipid metabolism, and vitamin metabolism. However, many aspects remain unclear, including the regulation of IAP expression and its effects on the expression of related genes. Therefore, we herein hypothesized that the transfection of an expression vector carrying the human ALPI gene, which encodes IAP, followed by a comprehensive gene expression analysis using RNA sequencing (RNA-seq), may suggest the effects of IAP on the expression of related genes and provide important evidence for elucidating the physiological functions of IAP.