The link between cutaneous inflammation and cognitive impairment.

Cognitive impairment is a symptom of neurological disorders, including dementia and Alzheimer's disease; and mild cognitive impairment can be a precursor of both disorders. Aged humans and animal models with other systemic disorders, such as cardiovascular diseases and diabetes, display a higher incidence of cognitive decline. Epidemiological studies have shown that the incidence of cognitive impairment also is higher in subjects with certain inflammatory skin disorders, including psoriasis and chronic eczematous dermatitis. Chronologically aged individuals exhibit increased cutaneous inflammation and elevated circulating cytokine levels, linked to alterations in epidermal function, which itself can induce cutaneous inflammation. Conversely, strategies that improve epidermal function can lower cytokine levels in both the skin and circulation. Thus, it seems likely that epidermal dysfunction could contribute, at least in part, to the development of chronic low-grade inflammation, also termed 'inflammaging', in the elderly. The evidence of cognitive impairment in patients with inflammatory dermatoses suggests a link between cutaneous inflammation and cognitive impairment. Because of the pathogenic role of epidermal dysfunction in ageing-associated cutaneous inflammation, improvements in epidermal function could be an alternative approach for mitigation of the ageing-associated decline in cognitive function.

CERKL is upregulated in cutaneous squamous cell carcinoma and maintains cellular sphingolipids and resistance to oxidative stress.

BACKGROUND: Ceramide kinase-like protein (CERKL) was originally described in retinal tissue. CERKL has been shown to protect cells from oxidative stress, and mutations in CERKL underlie the inherited disease retinitis pigmentosa. CERKL expression maintains cellular sphingolipids via an unknown mechanism. OBJECTIVES: To determine whether CERKL is expressed in epidermis and cutaneous squamous cell carcinoma (cSCC) and whether CERKL expression affects cSCC sphingolipid metabolism and susceptibility to oxidative stress. METHODS: CERKL expression was determined by RNA-Seq, quantitative polymerase chain reaction and immunohistochemistry. CERKL was knocked down in cSCC cells using small interfering RNA. Sphingolipid content was analysed by liquid chromatography-mass spectrometry. Oxidative stress was induced by treatment with H2 O2 , and apoptosis was measured using flow cytometry to determine annexin V binding. RESULTS: CERKL mRNA and protein are highly expressed in actinic keratosis and cSCC in comparison with normal epidermis. CERKL is also expressed in metabolically active epithelial cells in normal hair bulbs and sebaceous glands. CERKL knockdown in cultured cSCC cells reduces cellular sphingolipid content and enhances susceptibility to oxidative stress. CONCLUSIONS: These findings suggest that CERKL may be important in cSCC progression and could lead to novel strategies for prevention and treatment of cSCC.

CERKL is Upregulated in Cutaneous Squamous Cell Carcinoma and Maintains Cellular Sphingolipids and Resistance to Oxidative Stress.

BACKGROUND: Ceramide Kinase-Like Protein (CERKL) was originally described in retinal tissue. CERKL has been shown to protect cells from oxidative stress, and mutations in CERKL underlie the inherited disease, retinitis pigmentosa. CERKL expression maintains cellular sphingolipids via an unknown mechanism. OBJECTIVES: To determine whether CERKL is expressed in epidermis and cutaneous squamous cell carcinoma (cSCC) and whether CERKL expression affects cSCC sphingolipid metabolism and susceptibility to oxidative stress. METHODS: CERKL expression was determined by RNA-Seq, qPCR and immunohistochemistry. CERKL was knocked down in cSCC cells using siRNA. Sphingolipid content was analyzed by liquid chromatography-mass spectrometry (LC-MS). Oxidative stress was induced by treatment with H2 O2 , and apoptosis was measured using flow cytometry to determine annexin v binding. RESULTS: CERKL mRNA and protein are highly expressed in actinic keratosis and cSCC in comparison with normal epidermis. CERKL also is expressed in metabolically active epithelial cells in normal hair bulbs and sebaceous glands. CERKL knockdown in cultured cSCC cells reduces cellular sphingolipid content and enhances susceptibility to oxidative stress. CONCLUSIONS: These findings suggest that CERKL may be important in cSCC progression and could lead to novel strategies for prevention and treatment of cSCC.

Topical applications of an emollient reduce circulating pro-inflammatory cytokine levels in chronically aged humans: a pilot clinical study.

BACKGROUND: While increased levels of circulating inflammatory cytokines in chronologically aged humans have been linked to the development of ageing-associated chronic disorders (e.g., cardiovascular disease, type II diabetes, osteoporosis and Alzheimer's disease), approaches that reduce circulating cytokines are not yet available. In chronologically aged mice, we recently demonstrated that epidermal dysfunction largely accounts for age-associated elevations in circulating cytokine levels, and that improving epidermal function reduced circulating cytokine levels. OBJECTIVE: We performed a pilot study to determine whether improving epidermal function reduces circulating pro-inflammatory cytokine levels in aged humans. METHODS: Thirty-three aged humans were topically treated twice-daily for 30 days, with ≈ 3 mL of an emollient, previously shown to improve epidermal function, while untreated, aged humans and a cohort of young volunteers served as controls. Changes in epidermal function and levels of three key, age-related, plasma cytokines (IL-1ß, IL-6 and TNFα) were measured at baseline and after treatment, using Luminex 200™ system. RESULTS: We also found significantly higher baseline levels of IL-1ß, IL-6 and TNFα in aged vs. young humans (P < 0.001), as previously reported. Topical applications of the barrier repair emollient significantly enhanced epidermal permeability barrier function (P < 0.01) and stratum corneum hydration (P < 0.05). In parallel, circulating levels of IL-1ß and IL-6 normalized, while TNFα levels declined substantially. CONCLUSION: The results of this preliminary study suggest that a larger clinical trial should be performed to confirm whether improving epidermal function also can reduce circulating pro-inflammatory cytokine levels in aged humans, while also possibly attenuating the downstream development of chronic inflammatory disorders in the aged humans.

Endoplasmic reticulum Ca2+ depletion activates XBP1 and controls terminal differentiation in keratinocytes and epidermis.

BACKGROUND: Endoplasmic reticulum (ER) Ca(2+) depletion, previously shown to signal pathological stress responses, has more recently been found also to trigger homeostatic physiological processes such as differentiation. In keratinocytes and epidermis, terminal differentiation and barrier repair require physiological apoptosis and differentiation, as evidenced by protein synthesis, caspase 14 expression, lipid secretion and stratum corneum (SC) formation. OBJECTIVES: To investigate the role of Ca(2+) depletion-induced ER stress in keratinocyte differentiation and barrier repair in vivo and in cell culture. METHODS: The SERCA2 Ca(2+) pump inhibitor thapsigargin (TG) was used to deplete ER calcium both in cultured keratinocytes and in mice. Levels of the ER stress factor XBP1, loricrin, caspase 14, lipid synthesis and intracellular Ca(2+) were compared after both TG treatment and barrier abrogation. RESULTS: We showed that these components of terminal differentiation and barrier repair were signalled by physiological ER stress, via release of stratum granulosum (SG) ER Ca(2+) stores. We first found that keratinocyte and epidermal ER Ca(2+) depletion activated the ER-stress-induced transcription factor XBP1. Next, we demonstrated that external barrier perturbation resulted in both intracellular Ca(2+) emptying and XBP1 activation. Finally, we showed that TG treatment of intact skin did not perturb the permeability barrier, yet stimulated and mimicked the physiological processes of barrier recovery. CONCLUSIONS: This report is the first to quantify and localize ER Ca(2+) loss after barrier perturbation and show that homeostatic processes that restore barrier function in vivo can be reproduced solely by releasing ER Ca(2+), via induction of physiological ER stress.

Extracellular pH Controls NHE1 expression in epidermis and keratinocytes: implications for barrier repair.

We have previously shown that the Na+/H+ antiporter (NHE1) is an essential endogenous pathway responsible for stratum corneum (SC) acidification. Since the epidermis must re-establish its epidermal barrier after acute barrier perturbations, we asked whether the NHE1 was, in turn, regulated by changes in barrier status. We found that in vivo epidermal NHE1 expression was upregulated within hours of barrier disruption. We next asked whether NHE1 was regulated by barrier status per se, or by the SC alkalinization that accompanies barrier perturbation. NHE1 was upregulated by alkalinizing SC pH, whereas this antiporter was downregulated by acidifying SC pH, independent of changes in barrier status. Moreover, acidifying SC pH overrode the effects of barrier break in regulating NHE1 expression, suggesting that SC alkalinization is the major stimulus for increased NHE1 expression. Finally, we confirmed that the keratinocyte NHE1 antiporter is regulated by extracellular pH independent of barrier status, by demonstrating that NHE1 was upregulated in cultured keratinocytes exposed to pH 8.3 medium and downregulated in cultured keratinocytes exposed to pH 6.3 medium. These data suggest that the keratinocyte NHE1 is regulated by extracellular pH. SC barrier break also upregulates NHE1 expression, but this response seems to be mediated by concomitant changes in SC pH.

Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium influx and enhances cell differentiation.

Human epidermal keratinocytes synthesize, secrete, and degrade acetylcholine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetyl-choline. Because acetylcholine modulates transmembrane Ca2+ transport and intracellular metabolism in several types of cells, we hypothesized that cholinergic agents might have similar effects on keratinocytes. Nicotine increased in a concentration-dependent manner the amount of 45Ca2+ taken up by keratinocytes isolated from human neonatal fore-skins. This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinocyte nicotinic acetylcholine receptor(s). The sequences encoding the alpha 5 and alpha 7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes. These subunits, as well as the alpha 3, beta 2, and beta 4 subunits previously found in keratinocytes, can be components of Ca(2+)-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the rate of cell differentiation, we measured the nicotinic cholinergic effects on the expression of differentiation markers by cultured keratinocytes. Long-term incubations with micromolar concentrations of nicotine markedly increased the number of cells forming cornified envelopes and the number of cells staining with antibodies to suprabasal keratin 10, transglutaminase type I, involucrin, and filaggrin. The increased production of these differentiation-associated proteins was verified by Western blotting. Because nicotinic cholinergic stimulation causes transmembrane Ca2+ transport into keratinocytes, and because changes in concentrations of intracellular Ca2+ are known to alter various keratinocyte functions, including differentiation, the subcellular mechanisms mediating the autocrine and paracrine actions of epidermal acetylcholine on keratinocytes may involve Ca2+ as a second messenger.

Ion channels are linked to differentiation in keratinocytes.

In vivo and in vitro, keratinocyte differentiation is linked with increased extracellular Ca2+. In order to correlate ion channels with cell differentiation and investigate keratinocyte membrane responses to Ca2+, keratinocyte single channel currents were studied using the patch-clamp technique. The most frequently observed channel was a 14 pS nonspecific cation channel. This channel was permeable to Ca2+ and activated by physiological concentrations of Ca2+. We also found a 35 pS Cl- channel whose open probability increased with depolarization. Finally, a 70 pS K+ channel was seen only in cell-attached or nystatin-permeabilized patches. We correlated channel types with staining for involucrin, an early marker of keratinocyte differentiation. While the nonspecific cation channel and Cl- channel were seen in both involucrin positive and involucrin negative cells, all channels in which the K+ channel activity was present were involucrin positive. Membrane currents through these channels may be one pathway by which signals for keratinocyte proliferation or differentiation are sent.

Extracellular calcium affects the membrane currents of cultured human keratinocytes.

Electrophysiologic properties of cultured human keratinocytes were studied using the patch voltage-clamp technique. Undifferentiated, proliferative keratinocytes grown in low Ca2+ medium had an average resting membrane potential of -24 mV. Voltage-clamp experiments showed that these cells had two membrane ionic currents: a large voltage-independent leak conductance, and a smaller voltage-dependent Cl- current that activated with depolarization. Increasing the extracellular Ca2+ concentration from 0.15 to 2 mM resulted in a doubling of the magnitude of the voltage-gated current and a shift in current activation to more negative potentials. Since levels of extracellular Ca2+ can alter the morphology and differentiation state of keratinocytes, the finding of a Ca2(+)-activated Cl- current in these cells suggests a role for this conductance in the initiation of differentiation.