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.
Highlights
The skin is the interface between the human body and the external environment
We found that the different basal level of extracellular adenosine triphosphate (ATP) was attributed to trace amounts of fetal calf serum (FCS), but not to secreted cellular factors in the culture media (Figure S4)
We have shown that HaCaT cells spontaneously release and hydrolyze ATP
Summary
The skin is the interface between the human body and the external environment It prevents water loss and forms a defensive barrier against various harmful stimuli [1,2,3]. Keratinocytes are the predominant cells in the epidermis, and originate in the basal skin layer and grow outward through the epidermis to replace dead keratinocytes that constantly shed from the skin surface. Because of their primary function at the body-external environment interface, keratinocytes are prone to a variety of mechanical compressions and damage, which trigger wound healing, inflammation, and sensory stimuli transduction [9]. When the epidermal layer is compromised by a wound or injury, concentrated ATP is released from the damaged skin cells
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