Introduction of Alkaline Phosphatase
Alkaline phosphatase (AP, EC 3.1.3.1 orthophosphoric-monoesterase, alkaline optimum) is a membrane-bound enzyme found in nearly all living organisms. Mammalian APs exhibit low sequence identity with E. coli, but the conserved residues in the active site and ligands coordinating zinc atoms and magnesium ions suggest a similar catalytic mechanism in prokaryotic and eukaryotic APs. Human APs, coded by up to four genes, consist of four isoenzymes: intestinal alkaline phosphatase (IAP), placental alkaline phosphatase (PLAP), germ cell alkaline phosphatase (GCAP), and tissue nonspecific alkaline phosphatase (TNAP). TNAP has three isoforms: bone, liver, and kidney TNAP. In mice, there are three tissue-specific APs (intestinal, intestinal-like, embryonic) and one tissue-nonspecific AP. TNAP is a key focus in stem cell biology, while embryonic AP (EAP) and GCAP cannot be overlooked due to their expression in pluripotent inner cell mass and primordial cells. TNAP and EAP/GCAP are associated with germ layers, progenitors, and undifferentiated cells, while tissue-specific APs (TSAPs) express as cells undergo differentiation and maturation in specific lineages and tissues, reflecting their connection to cellular functions.
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Fig. 1 Intestinal alkaline phosphatase (IALP) (Santos G. M., et al. 2022).
Expression of AP during Development
AP expression during development aligns with stem cell precursors and niches. In mice, AP is detected in 2-cell preimplantation embryos, expressed in each embryonic cell until the early blastocyst stage. Initially expressed by trophectoderm and inner cell mass (ICM), late blastocyst stages show strict ICM expression. The isoenzyme EAP is described in the preimplantation stage, with TNAP expressed 10 times less. Around 7 days post coitum (dpc), EAP expression decreases, and TNAP becomes the major gene expressed in primordial germ (PG) cells between 7-14 dpc. In humans, alkaline phosphatases are detectable prior to 4 weeks of gestation. Human migrating PG cells exhibit GCAP activity, primarily synthesized in the testes, cervix, and thymus in adults. TNAP expression in mice is observed in the neuroectoderm (8th dpc) and later in the brain, spinal cord, mesencephalon, rhombencephalon, and cranial nerves (9.5-14.5 dpc). TNAP is involved in nervous system development, acting as an ectonucleotidase in neurogenic zones or interacting with collagen during neuronal migration. TNAP activity remains detectable in the choroid plexus up to adult age. In vertebrates, TNAP is dominant in the developing skeleton, and crucial for tissue mineralization in chondrocytes and osteoblasts. Another AP, TSAP, is associated with cell differentiation, serving as a marker for this process. The dynamic expression patterns of AP isoforms contribute to the intricate processes of tissue development and differentiation.
Role and Function of AP
AP serves a pivotal role through its catalytic activities, including hydrolytic, phosphotransferase, and pyrophosphatase functions. While the hydrolytic activity is a common reaction, individual APs exhibit unique functions, evident from organisms lacking functional AP. TNAP plays a critical role in hard tissue mineralization and vitamin B metabolism, influencing bone matrix formation and neurotransmitter metabolism. Although ethical constraints limit experimental data on human primordial germ cells (PGC), similarities with mouse PGC suggest a comparable state for GCAP. In primates, PLAP facilitates maternal IgG transport and enhances embryonic growth. TNAP's multifaceted functions extend to the mineralization of hard tissue and vitamin B metabolism, essential for bone health and nervous system development. IAP contributes to fatty acid transport, regulates triglycerides, modulates pH in the duodenum, and detoxifies bacterial endotoxins in the colon. The diverse functions of AP highlight its significance in various physiological processes, emphasizing its importance in maintaining overall health and development.
AP and Stem Cells
AP plays a crucial role in stem cell biology, with its activity regulated by the microenvironment, making it a valuable marker for differentiation processes. In this context, TNAP has garnered significant attention, especially in the realm of pluripotent stem cells.
Pluripotent stem cells, such as embryonic stem cells (ES), embryonal cancer cells (EC), and induced pluripotent stem cells (iPS), exhibit elevated levels of AP activity. TNAP, in particular, is upregulated during the reprogramming of somatic cells into iPS cells. This underscores the dynamic nature of AP expression and its association with the developmental status of cells or tissues.
The regulation of AP expression is finely tuned by the microenvironment, reflecting the intricate balance required for cellular differentiation. AP activity is considered a reliable marker for pluripotent stem cells, demonstrating its importance in identifying and characterizing these cells. Its maintenance has been correlated with the clonogenic and self-renewal potential of undifferentiated human ES cells, emphasizing its significance in maintaining the pluripotent state.
However, the role of AP in various stem cell populations remains a subject of ongoing investigation. In mesenchymal stem cells (MSC), TNAP is acknowledged as a marker of stemness in some populations, highlighting its potential utility in identifying undifferentiated cells. Yet, in other adult stem cells, TNAP expression appears to be more closely associated with differentiation processes rather than the maintenance of stemness.
In summary, while TNAP expression serves as a suitable marker for pluripotent stem cells, its specific significance and role in different stem cell populations are not yet fully understood. The complex regulatory networks governing AP expression in stem cell biology warrant further exploration. Researchers must delve into the intricacies of these networks to unravel the functional implications of AP in various stem cell types.
References
- Štefková K., et al. Alkaline phosphatase in stem cells. Stem Cells International. 2015, 2015.
- Santos G. M., et al. Intestinal alkaline phosphatase: A review of this enzyme role in the intestinal barrier function. Microorganisms. 2022, 10(4): 746.
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