Y-27632 preserves epidermal integrity in a human skin organ-culture (hSOC) system by regulating AKT and ERK signaling pathways.

BACKGROUND

The human skin organ culture (hSOC) developed a century ago has been widely used to study various aspects of human skin development, differentiation, function, disease as well as skin appendages biology, however, maintaining the integrity of epidermal structure in long-term culture, has remained a challenge.

OBJECTIVES

Here we tried to establish a culture system using supplemented William's E medium in the presence of a ROCK inhibitor Y-27632 to maintain epidermal architecture in the long-term hSOC and to investigate the underlying mechanisms.

METHODS

Human breast skins, cut into 5 mm × 5 mm pieces, were cultured in supplemented William's E medium in the presence of 30μM Y-27632. The cultured skin tissues were collected at different time points for analysis of epidermal cell proliferation and differentiation by real time qRT-PCR and immunofluorescence (IF) staining. The keratinocyte suspension assay and in vivo treatment of Y-27632 on mouse were also carried out to study that the regulation of Y-27632 on keratinocyte proliferation and differentiation.

RESULTS

We found Y-27632 not only enhanced both basal cell proliferation and expression of suprabasal cell differentiation markers, but also maintained the balance of keratinocyte proliferation and differentiation through activation of AKT pathways on one hand and inhibition of ERK pathways on the other hand. The AKT inhibitor MK-2206 blocked the epidermal preservation effect of Y-27632, while the MEK/ERK inhibitor U0126 enhanced the preservation of epidermal structure in the hSOC.

CONCLUSIONS

Y-227632 can maintain skin epidermal integrity through regulation of AKT and ERK activity in the hSOC.