The life in a gradient: calcium, the lncRNA SPRR2C and mir542/mir196a meet in the epidermis to regulate the aging process

Abstract

The turnover of the epidermis beginning with the progenitor cells in the basal layer to the fully differentiated corneocytes is tightly regulated by calcium. Calcium more than anything else promotes the differentiation of keratinocytes which implies the need for a calcium gradient with low concentrations in the stratum basale and high concentrations in the stratum granulosum. One of the hallmarks of skin aging is a collapse of this gradient that has a direct impact on the epidermal fitness. The rise of calcium in the stratum basale reduces cell proliferation, whereas the drop of calcium in the stratum granulosum leads to a changed composition of the cornified envelope. We showed that keratinocytes respond to the calcium induced block of cell division by a large increase of the expression of several miRNAs (hsa-mir542-5p, hsa-mir125a, hsa-mir135a-5p, hsa-mir196a-5p, hsa-mir491-5p and hsa-mir552-5p). The pitfall of this rescue mechanism is a dramatic change in gene expression which causes a further impairment of the epidermal barrier. This effect is attenuated by a pseudogene (SPRR2C) that gives rise to a lncRNA. SPRR2C specifically resides in the stratum granulosum/corneum thus acting as a sponge for miRNAs.

Keywords: epidermal barrier; lncRNA; miRNA; pseudogene; skin aging.

Figure 1

Age dependent expression of SPRR2C and conservation in evolution. In (A) the ΔΔCt values of SPPR2C in foreskin samples of middle aged (age range: 24-39 years) and elderly people (age range: 60 to 76 years) are shown after normalization to young foreskin samples (age range: 2.5-8 years), (N=5 in each group;). One-way ANOVA analysis indicates a significant difference between groups (p= 0.0153285). In (B) a multiple sequence alignment (first 54 nucleotides of the ORF) of SPRR2C in hominids, apes and mammals was calculated using MVIEW (https://www.ebi.ac.uk/Tools/msa/mview/). In most hominids the 6^th codon (CAG) encodes a glutamine but is changed into a STOP codon (TAG) in Homo sapiens, Pan troglodytes and Pan paniscus. 1: Homo sapiens; 2: Pan troglodytes; 3: Pan paniscus; 4: Gorilla gorilla gorilla; 5: Pongo abelii; 6: Sapajus paella; 7: Papio Anubis; 8: Cebus capucinus; 9: Chlorocebus sabaeus; and 10: Condylura cristata.

Figure 2

The interaction between lncRNA SPRR2C and several miRNAs. In (A) the SPRR2C-miRNA hybrids are shown. The SPRR2C region is presented in red, the appropriate miRNA in green. Both RNA-miRNA hybrids and mfe were calculated using RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid). In (B) the SPRR2C mRNA is presented and the binding sites for hsa-mir542-5p, hsa-mir125a, hsa-mir135a-5p, hsa-mir196a-5p, hsa-mir491-5p and hsa-mir552-5p are marked. hsa-mir542-5p: red letters; hsa-mir125a-5p: yellow letters; hsa-mir135a-5p: red bracket; hsa-mir196a-5p: blue bracket; hsa-mir491-5p: green letters; hsa-mir552-5p: lower case letters.

Figure 3

miRNA expression levels during aging. RT-PCR analysis of miRNAs specifically targeting SPRR2C. The ΔΔCt values for hsa-mir542-5p, hsa-mir125a-5p, hsa-mir135a-5p, hsa-mir196a-5p, hsa-mir491-5p and hsa-mir552-5p are presented. All values are normalized to young foreskin (age range: 2.5-8 years). Black columns: middle aged foreskin samples (age range: 24-39 years); grey columns: old foreskin samples (age range: 60 to 76 years). (N=5 in each group; *: p<0.1; **: p< 0.05; ***: p<0.01).

Figure 4

The binding of mir542-5p to its target SPRR2C. A dual luciferase assay was performed by sequentially measuring firefly and renilla luciferase. A immortalized keratinocyte cell line was transfected with 350 ng psiCHECK2™-SPRR2C (281-486) and either 125 nM MISSION miRNA MIMIC hsa-mir542 or MISSION miRNA MIMIC Negative Control #1. Black bars indicate a co-transfection with mir-542-5p, grey bars a co-transfection with the control miRNA. All data are normalized to the firefly luciferase activity (N=4). An independent t-test analysis indicated a significant difference.

Figure 5

The role of the lncRNA SPRR2c and hsa-mir542-5p in keratinocyte differentiation. RT-PCR analysis of selected CE family members are presented as ΔΔCt values in (A, B). In (A) SPRR2C (pLL3.7-SPRR2c) is overexpressed after transduction of HaCaT cells with lentiviral particles. In (B) the increase of expression of several CE family members is analyzed after addition of 0.8 mM CaCl₂ and transfection of HaCaT cells with hsa-mir542a-5p (N=4; *: p<0.1; **: p<0.05; ***: p<0.01).

Figure 6

The doubling time of HaCaT cells in dependence of calcium and mir542-5p. HaCaT cells were treated with 0.5, 0.8 and 1 mM CaCl2 to induce the formation of the cornified envelope. In parallel cells were transfected with a control miRNA and hsa-mir542-5p. Cell numbers were calculated 24, 48 and 72 hours after start of treatment (N=4-6; *: p<0.1; **: p<0.05; ***: p<0.01).

Figure 7

ISH staining of SPRR2c and mir542-5p. (A–C) ISH staining of SPRR2C. (A) Foreskin sample from a 6-year-old; (B) Foreskin sample of a 30-year-old; (C) Foreskin sample of a 61 year old. Increased SPRR2C levels were detected in old skin samples compared to young skin. (D–F) ISH staining of mir542-5p. (D) Foreskin sample of an eight-year old; (E) Foreskin sample of a 30-year-old; (F) Foreskin sample of a 61-year old. During aging the hsa-mir542-5p levels gradually increase. (B, E) as well as (C, F) are specimen from identical donors. The intensity of the black-purple staining correlates with the amount of either SPRR2C or mir-542-5p. Stratum basale appears brownish due to the presence of melanocytes and is no specific staining. Scale bar: 50 μm.

Figure 8

The interplay between the lncRNA SPRR2c and several miRNAs. (A) represents the situation in young and healthy skin. (D) is representative for aged skin. (B, C) are hypothetical situations with either the absence of miRNA (B) or SPRR2C (C). Green triangle: epidermal calcium gradient; green box: evenly distributed calcium after the collapse of the epidermal calcium gradient (e.g. during skin aging). Red circle: Cell cycle. Size indicates the cell proliferation rate (the bigger the faster). Brown box: cornified envelope (the intensity is correlated with the amounts of proteins in the CE). miRNAs: blue; lncRNA: yellow.