Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 Modulates Local ATP-Induced Calcium Signaling in Human HaCaT Keratinocytes

Abstract

Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP) as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP) accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

NTPDase2 and Purinergic Signaling Control Progenitor Cell Proliferation in Neurogenic Niches of the Adult Mouse Brain

“…Deletion or knockdown of Entpd2 was previously shown to reduce extracellular ATP hydrolysis and to increase extracellular ATP concentrations . Similarly, inhibition of ectonucleotidase activity by the NTPDase inhibitor PV4 in rat striatum resulted in an increase of resting extracellular ATP concentrations from 40 to 360 nM .…”

Transmission of mechanical information by purinergic signaling

“…ATP degradation half-time in the extracellular space was 20 min (95% CI: 17 to 25) for C2-OB cells, compared to ~10 min by primary rat osteoblasts (46), and ~5 min by primary murine osteoblasts (47). However, in osteoblasts ATP degradation was too slow to affect purinergic transmission initiated by a single cell, contrary to other cell types in which paracrine responses following single cell mechanical stimulation were potentiated when ecto-nucleotidases were inhibited, such as in bovine endothelial cells treated with ARL-67156 (48,49), or in human keratinocytes with siRNA-silenced NTPDase2 (50). Nevertheless, since most ecto-nucleotidases have a micromolar affinity for ATP (40), low hydrolysis rates were expected for the amounts of ATP released by a single mechanically stimulated osteoblast.…”

Zinc and skin biology

“…2). Thus, CD39 is so far considered to be responsible for ecto-NTPDase activity in the epidermis [50,54]. Although epidermal cells prepared from the ear skin of ZA mice contained a normal contingent of LCs, the number of epidermal LCs in mice fed a ZD diet decreased with time on the diet, and few LCs were observed after 6 weeks.…”