Osmolyte transporter expression is reduced in photoaged human skin: Implications for skin hydration in aging

Abstract Aging is characterized by the deterioration of tissue structure and function. In skin, environmental factors, for example, ultraviolet radiation (UVR), can accelerate the effects of aging such as decline in barrier function and subsequent loss of hydration. Water homeostasis is vital for all cellular functions and it is known that organic osmolyte transport is critical to this process. Therefore, we hypothesized that as we age, these tightly controlled physiological mechanisms become disrupted, possibly due to loss of transporter expression. We investigated this in vivo, using human skin samples from photoprotected and photoexposed sites of young and aged volunteers. We show a reduction in keratinocyte cell size with age and a downregulation of osmolyte transporters SMIT and TAUT with both chronic and acute UVR exposure. Single‐cell live imaging demonstrated that aged keratinocytes lack efficient cell volume recovery mechanisms possessed by young keratinocytes following physiological stress. However, addition of exogenous taurine significantly rescued cell volume; this was corroborated by a reduction in TAUT mRNA and protein in aged, as compared to young, keratinocytes. Collectively, these novel data demonstrate that human epidermal keratinocytes possess osmolyte‐mediated cell volume regulatory mechanisms, which may be compromised in aging. Therefore, this suggests that organic osmolytes—especially taurine—play a critical role in cutaneous age‐related xerosis and highlights a fundamental mechanism, vital to our understanding of the pathophysiology of skin aging.

. BGT-1 and HMIT protein expression in young and aged, photoprotected and photoexposed human skin.
Immunofluorescence for (a-d) BGT-1 and (f-i) HMIT in and aged photoprotected and photoexposed skin. Immunofluorescence intensity analysis showed (e) BGT-1 protein expression is downregulated in aged photoexposed skin compared to young photoexposed skin (p=0.0206). (j) HMIT protein expression did not change with age and photoexposure. Data expressed as mean ± SD (two-way ANOVA), young n=5, aged n=6. Scale bars = 20μm. BGT-1, betaine transporter; HMIT, hydrogencoupled myoinositol transporter.   Young and aged NHEKs exposed to hyperosmotic conditions in the absence or presence of organic osmolytes were captured using single cell live imaging. Cell images were assessed for (a-b) percentage cell volume regained and (c-d) maximum cell volume decrease. Data expressed as mean ± SD (one-way ANOVA), n=10 cells per donor from 3 young and 3 aged donors. NHEKs, normal human epidermal keratinocytes.

Table S1. Donor details for human skin studies.
Human skin samples were collected from healthy volunteers recruited into two different cohorts. (a) Cohort one included 5 young (mean age 27.8 years) and six aged (mean age 69.5 years) donors. (b) Cohort two included 5 individuals with a mean age 36.4 years.

Study cohorts and protocols
Cohort one (ref#15464) included five young (18-30 years; 3 female and 2 male) and six aged (>65 years; 3 female and 3 male) volunteers. Each volunteer had two skin biopsies taken, one from photoprotected buttock and one from photoexposed forearm. For cohort two (ref#15439), five volunteers (average age 37.25 years; 1 female and 4 male) were exposed to acute solar simulated radiation (SSR) at eight times standard erythema dose (SED; 80 mJ) at the photoprotected buttock site. A skin biopsy was taken from an unirradiated site (baseline) then at 1-, 3-and 72-hours post-SSR exposure.

Immunofluorescence
For all immunofluorescence carried out for the organic osmolyte transporters (single or dual stains) sections were air dried for 10 minutes at room temperature (RT) and fixed in chilled 100% acetone for 15 minutes. For single immunofluorescence of e-cadherin, sections were fixed in chilled 4% PFA for 20 minutes. Blocking was carried out using 2.5% normal horse serum for 30 minutes at RT. Tris-buffered saline solution was used for all wash steps.

Protein extraction
NHEKs were grown in a 12-well plate until confluency for protein extraction. Total protein was isolated from NHEKs using extraction buffer of the following composition in 100ml of distilled water: 120mM sodium chloride, 25mM HEPES, 1% triton X-100, 25mM sodium fluoride, 1mM sodium orthovanadate, 0.2% SDS and 0.1mg/ml protease inhibitor cocktail (catalogue #P8340; Sigma). Cells were washed twice with ice cold PBS then 100μl of extraction buffer was added to each well and cell scraping was used to collect the cells in the buffer. The lysate was then kept on ice for 30 minutes before sonication at half maximum speed for 5 seconds. Samples were then centrifuged at 13000rpm for 20 minutes and the supernatant was collected. Protein concentration was quantified using the Invitrogen Qubit 4 Fluorometer (ThermoFisher Scientific).