Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8

Hitomi Fujisaki; Sugiko Futaki; Masashi Yamada; Kiyotoshi Sekiguchi; Toshihiko Hayashi; Takashi Ikejima; Shunji Hattori

doi:10.1016/j.reth.2018.04.001

Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8

Regen Ther. 2018 May 24:8:73-79. doi: 10.1016/j.reth.2018.04.001. eCollection 2018 Jun.

Authors

Hitomi Fujisaki¹, Sugiko Futaki², Masashi Yamada³, Kiyotoshi Sekiguchi³, Toshihiko Hayashi⁴, Takashi Ikejima⁴, Shunji Hattori¹

Affiliations

¹ Nippi Research Institute of Biomatrix, Toride, Ibaraki, Japan.
² Department of Anatomy and Cell Biology, Osaka Medical College, Osaka, Japan.
³ Division of Matrixome Research and Application, Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
⁴ China-Japan Research Institute of Medical and Pharmaceutical Sciences, Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, China.

Abstract

Keratinocyte line cells HaCaT and FEPE1L-8 are used for skin model with type I collagen fibrils (gels). For this purpose, not only differentiation but also regulation of proliferation on type I collagen gels by exogenous calcium concentration is important. When exogenous calcium concentration is low, primary keratinocyte proliferation is repressed and eventually cells are induced to apoptosis on type I collagen gels. The apoptosis induced on type I collagen gels is suppressed by increasing calcium concentration in the medium. That is, higher exogenous calcium concentration is necessary for primary keratinocyte survival on type I collagen gels than for that on dish surface culture. Meanwhile much higher exogenous calcium causes cell differentiation and inhibition of proliferation. The optimal calcium concentrations for proliferation on type I collagen gels have not been clarified in keratinocyte line cells. HaCaT cells have a unique calcium sensitivity in comparison with primary keratinocytes, whereas FEPE1L-8 cells have a similar sensitivity to primary keratinocytes. In this study, we compared the effect of calcium concentrations on proliferation of HaCaT and FEPE1L-8 cells on type I collagen gels. On type I collagen gels, both line cells required higher calcium concentrations for proliferation than on dish surface. HaCaT cells proliferated better in a wider range of calcium concentrations than FEPE1L-8 cells.

Keywords: Calcium concentration; DAG, diacylglycerol; DMEM (0), DMEM supplemented without fetal bovine serum; DMEM (10), DMEM supplemented with 10% fetal bovine serum; DMEM, Dulbecco's Modified Eagle's Medium; ECM, extracellular matrix; HBSS, Hanks' balanced salt solution; HEPES, 4-(2-hydroxyethyl-1-piperazineethanesulfonic acid; IP3, inositol trisphosphate; K110, K110 type II medium; Keratinocyte proliferation; MTT, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide; PI, propidium iodide; PI3K, phosphoinositide 3-OH-kinase; PIP2, hydrolyze phosphatidylinositol bisphosphate; PKC, protein kinase C; Type I collagen gel; WST-8, (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt.