Calcium enhances mouse keratinocyte differentiation in vitro to differentially regulate expression of papillomavirus authentic and codon modified L1 genes

Abstract

Here, we first wished to establish for mouse primary keratinocytes (KCs) the Ca(2+) concentrations that were associated with KC differentiation in vitro. Using the range of Ca(2+) concentrations (0-6 mM) to differentiate primary KCs in culture to varying extents for 2 days, we then examined how KC differentiation impacted on expression of papillomavirus (PV) native (Nat) and codon modified (Mod) L1 genes. L1 mRNAs transcribed from either Nat or Mod L1 genes were present in similar amounts in KCs exposed to six Ca(2+) concentrations. However, expression of the L1 proteins from two Mod L1 genes were down-regulated, with no L1 signal detected in KCs exposed to 6 mM Ca(2+). In contrast, L1 proteins expressed from the two Nat L1 genes were not detectable in KCs without Ca(2+), but dramatically up-regulated as the KC cultures exposed to Ca(2+) from 0.5 to 2 mM, then down-regulated in KCs exposed to Ca(2+) from 4 to 6 mM. The different regulatory roles of the Ca(2+) in L1 protein expression from Nat and Mod L1 genes in cultured KCs were confirmed by TGF-beta1 experiments. We observed that aminoacyl-tRNAs (aa-tRNAs) from the 2 mM Ca(2+)-treated KCs only significantly enhanced the Nat L1 mRNAs translation in vitro, suggesting that aa-tRNAs play a differentially regulatory role in translations of the PV Nat and Mod L1 mRNAs. Importantly, the Ca(2+) experimental model provides evidence that mouse primary KCs could be transiently infected by BPV1 virus to express L1 mRNA and protein, which is very useful for future HPV virus infection study.

β-Adducin siRNA disruption of the spectrin-based cytoskeleton in differentiating keratinocytes prevented by calcium acting through calmodulin/epidermal growth factor receptor/cadherin pathway

“…K14 expression was obviously enhanced in β-adducin-siRNA transfected keratinocytes without Ca 2+ treatment, while Ca 2+ treatment prevented the up-regulation of the K14 induced by β-adducinsiRNA transfection in keratinocytes (Fig. 5), consistent with our previous results that Ca 2+ down-regulated the expression of K14 in mouse primary keratinocytes [18]. Similar to our previous study, the expression of tubulin in both control and β-adducin-siRNA transfected keratinocytes without Ca 2+ treatment was distinctly higher than that in those cells with Ca 2+ treatment (Fig.…”

“…Both K1 and K10 among the keratin family are specifically expressed in the spinous and granular layers of the epidermis, which are recognized as the markers of keratinocyte differentiation [26]. During differentiation, the keratinocytes undergo a shape change from small and round to large and flat by expressing several differentiation markers including involucrin and keratin 10 [3,18]. Thus, together with our previous results, the present data suggest that the expression of β-adducin regulated by Ca 2+ is necessary for keratinocyte differentiation, implying further that β-adducin may play an important role in cell differentiation leading to the development of the epidermis and skin.…”

“…The existence of the Ca 2+ gradient reveals its importance as a physiological regulator of epidermal development and differentiation [12]. Furthermore, as an enhancer of cell differentiation, Ca 2 + promotes mouse keratinocyte differentiation in vitro, which regulates the expression of differentiation markers such as involucrin and K1/K10 and viral capsid proteins [18]. Ca 2 + also plays roles in directional sensing, cytoskeleton redistribution, traction force generation, and relocation of focal adhesions in migrating cells [15,19].…”

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

“…It has been reported that an intermediate level of calcium (0.1- 0.16 mM) substantially induced the expression of early differentiation markers, such as K1 and K10, during the first 24 h upon calcium switch, whereas K1 and K10 was only induced in a small fraction of cells when treated with higher calcium medium (1 mM) 19,20 . However, the higher calcium medium (≥1 mM) has been widely used in many other studies to rapidly and robustly induce KC stratification and the expression of late differentiation genes, such as INV and LOR 4,21,22,23 . Based on our experience, an intermediate level of calcium, such as 0.2 mM CaCl 2 , leads to a gradual and slower onset of terminal differentiation, whereas a higher calcium levels (≥1 mM) drastically speed up the onset of KC terminal differentiation, leading to a shorter time window to study the cellular changes during differentiation.…”

Expression of HPV 58 Long and Short L1 Capsid Proteins in Primary Mouse Keratinocyte Cultures

“…We demonstrated in vitro and in vivo that gene codon composition plays a role in differentiation-dependent expression of L1 proteins in KCs [21]. Using the KC culture system, we also proved that calcium enhances both mouse and human KC differentiation to differentially regulate expression of PV authentic and codon modified L1 ORFs [22]. Furthermore, generalised substitution of isoencoding codons shortens the duration of PV L1 protein expression in transiently L1-transfected KCs due to cell differentiation [23].…”