THE VOLUME-REGULATED ANION CHANNEL PROTEIN LRRC8A FOR USE IN ALTERING EPIDERMAL KERATINOCYTE DIFFERENTIATION
20200393448 · 2020-12-17
Inventors
Cpc classification
G01N33/5008
PHYSICS
C12N9/22
CHEMISTRY; METALLURGY
A61K8/64
HUMAN NECESSITIES
International classification
G01N33/50
PHYSICS
A61K8/64
HUMAN NECESSITIES
Abstract
The present invention relates to the leucine-rich repeat-containing protein 8A (LRRC8A), and/or an activator of LRRC8A, for use in the treatment and/or prevention of a skin condition associated with an altered differentiation of keratinocytes. Preferably, the skin condition associated with an altered differentiation of keratinocytes is psoriasis or dermatitis, preferably atopic dermatitis. The present invention further relates to a method of identifying a compound capable of altering the differentiation of keratinocytes, the method comprising the steps of (a) contacting keratinocytes with a test compound and determining the amount of LRRC8A protein or LRRC8A transcript in said keratinocytes; and (b) comparing the amount of LRRC8A protein or LRRC8A transcript determined in step (a) with the amount of LRRC8A protein or LRRC8A transcript in a control not contacted with said test compound, wherein a change in the amount of LRRC8A protein or LRRC8A transcript after contacting the keratinocytes with the test compound indicates that the test compound is capable of altering the differentiation of keratinocytes. Furthermore, the present invention relates to a method of identifying a compound capable of altering the differentiation of keratinocytes, the method comprising the steps of (a) contacting keratinocytes with a test compound and determining the activity of (a) VRAC(s) comprising LRRC8A in said keratinocytes; and (b) comparing the activity determined in step (a) with the activity in a control not contacted with said test compound, wherein a change in the activity of (a) VRAC(s) comprising LRRC8A after contacting the keratinocytes with the test compound indicates that the test compound is capable of altering the differentiation of keratinocytes. The present invention further relates to an inhibitor of the leucine-rich repeat-containing protein 8A (LRRC8A) for use in the treatment and/or prevention of a skin condition selected from skin injury and impaired wound healing, as well as to a cosmetic method for alleviating the effects of a skin condition on the appearance of the skin of an affected individual, the method comprising topically administering an effective amount of (i) leucine-rich repeat-containing protein 8A (LRRC8A); (ii) an activator of LRRC8A; (iii) LRRC8A and an activator of LRRC8A; or (iv) an inhibitor of LRRC8A.
Claims
1. The leucine-rich repeat-containing protein 8A (LRRC8A), and/or an activator of LRRC8A, for use in the treatment and/or prevention of a skin condition associated with an altered differentiation of keratinocytes.
2. The LRRC8A and/or the activator for use according to claim 1, wherein the skin condition associated with an altered differentiation of keratinocytes is a condition characterised by enhanced epidermal proliferation.
3. The LRRC8A and/or the activator for use according to claim 1, wherein the skin condition associated with an altered differentiation of keratinocytes is psoriasis or dermatitis, preferably atopic dermatitis.
4. The activator of LRRC8A for use according to claim 1, wherein the activator is (i) a vector encoding, in expressible form, LRRC8A; or (ii) a regulator of gene expression that up-regulates the expression of endogenously present LRRC8A.
5. The activator of LRRC8A for use according to claim 4 (ii), wherein the regulator of gene expression that up-regulates the expression of endogenously present LRRC8A is selected from (i) CRISPR-Cas9-based regulators; (ii) CRISPR-Cpf1-based regulators; (iii) programmable sequence-specific genome editing nucleases selected from zinc-finger nucleases (ZNFs) and transcriptional activator-like effector nucleases (TALENs); (iv) meganucleases; (v) small molecules; (vi) antibodies or antibody mimetics; (vii) aptamers; and (viii) inhibitory nucleic acid molecules selected from siRNA, shRNA, miRNA, ribozymes and antisense nucleic acid molecules.
6. A method of identifying a compound capable of altering the differentiation of keratinocytes, the method comprising the steps of (a) contacting keratinocytes with a test compound and determining the amount of LRRC8A protein or LRRC8A transcript in said keratinocytes; and (b) comparing the amount of LRRC8A protein or LRRC8A transcript determined in step (a) with the amount of LRRC8A protein or LRRC8A transcript in a control not contacted with said test compound, wherein a change in the amount of LRRC8A protein or LRRC8A transcript after contacting the keratinocytes with the test compound indicates that the test compound is capable of altering the differentiation of keratinocytes.
7. A method of identifying a compound capable of altering the differentiation of keratinocytes, the method comprising the steps of (a) contacting keratinocytes with a test compound and determining the activity of (a) VRAC(s) comprising LRRC8A in said keratinocytes; and (b) comparing the activity determined in step (a) with the activity in a control not contacted with said test compound, wherein a change in the activity of (a) VRAC(s) comprising LRRC8A after contacting the keratinocytes with the test compound indicates that the test compound is capable of altering the differentiation of keratinocytes.
8. The method of claim 6 or 7, further comprising determining the expression level of at least one marker selected from keratin 1 (KRT1), keratin 10 (KRT10), involucrin (IVL), filaggrin (FLG), loricrin (LOR), keratin 4 (KRT4), keratin 15 (KRT15), transglutaminase 1 (TGM1), S100 calcium binding protein A7 (S100A7), S100 calcium binding protein A8 (S100A8), S100 calcium binding protein A9 (S100A9), C-X-C motif chemokine ligand 1 (CXCL1), C-X-C motif chemokine ligand 8 (CXCL8), small proline rich protein 2C (SPRR2C), small proline rich protein 2D (SPRR2D), serpin family B member 3 (SERPINB3), serpin family B member 4 (SERPINB4), peptidase inhibitor 3 (PI3), lipocalin 2 (LCN2), keratin 6A (KRT6A), keratin 16 (KRT16), beta-defensin 1 (DEFB1) and marker of proliferation Ki-67 (MK167)
9. The method of claim 6, wherein an increase in the amount of LRRC8A protein or LRRC8A transcript after contacting the keratinocytes with the test compound and/or an increase in the activity of (a) VRAC(s) comprising LRRC8A after contacting the keratinocytes with the test compound indicates that the test compound is a compound suitable for use in the treatment and/or prevention of a skin condition associated with an altered differentiation of keratinocytes.
10. The method of claim 9, wherein the skin condition associated with an altered differentiation of keratinocytes is psoriasis or dermatitis, preferably atopic dermatitis.
11. An inhibitor of the leucine-rich repeat-containing protein 8A (LRRC8A) for use in the treatment and/or prevention of a skin condition selected from skin injury and impaired wound healing.
12. The inhibitor for use according to claim 11, wherein (i) the inhibitor decreases the expression of LRRC8A; and/or (ii) the inhibitor decreases the activity of volume-regulated anion channels (VRACs) comprising LRRC8A.
13. The LRRC8A and/or the activator for use according to claim 1, wherein the LRRC8A and/or the activator is comprised in a pharmaceutical composition.
14. The method of claim 6, wherein a decrease in the amount of LRRC8A protein or LRRC8A transcript after contacting the keratinocytes with the test compound and/or a decrease in the activity of (a) VRAC(s) comprising LRRC8A after contacting the keratinocytes with the test compound indicates that the test compound is a compound suitable for use in the treatment and/or prevention of a skin condition selected from skin injury and impaired wound healing.
15. A cosmetic method for treating the skin of an individual, the method comprising topically administering an effective amount of (i) leucine-rich repeat-containing protein 8A (LRRC8A); (ii) an activator of LRRC8A; (iii) LRRC8A and an activator of LRRC8A; or (iv) an inhibitor of LRRC8A.
16. The inhibitor for use according to claim 11, wherein the inhibitor is comprised in a pharmaceutical composition.
Description
[0194] The figures show:
[0195]
[0196] (A) RT-PCR analysis using total RNA isolated from HaCaT keratinocytes and normal human epidermal keratinocytes (NHK) revealed specific PCR products for all LRRC8 gene family members (LRRC8A-E). -Actin-specific PCR product was obtained and served as loading control. No PCR product was obtained when reverse transcriptase (RT) was omitted. M, Molecular weight standard. (B) Western blot analysis using whole cell extracts of normal human epidermal keratinocytes (NHK) and HaCaT keratinocytes showed a strong LRRC8A antibody signal at around 100 kDa, which is close to the calculated molecular mass of 94 kDa. -Actin-specific antibody signal was obtained and served as loading control. M, Molecular weight standard. (C) Immunohistochemistry analysis using LRRC8A antibody revealed the existence of LRRC8A protein in human skin biopsies. Localization of LRRCA8 was visualized by using primary Anti-LRRC8A antibody and FITC-labeled secondary antibody. Green fluorescent FITC signal was preferentially detected in basal epidermal keratinocytes and declined towards the outer keratinocyte layers. Isotype antibody control showed no green fluorescent signal confirming specificity of the LRRC8A antibody. DNA was counterstained with DAPI to identify the cell nucleus. Note: The green fluorescent FITC signal is shown in white/light grey and DAPI is shown in dark grey in this picture.
[0197]
[0198] Western blot (A) and immunofluorescence analysis (B) of HaCaT cells at different stages of differentiation showed that LRRC8A protein level (A) as well as membrane localization of LRRC8A (B) is first increased before it reached its maximum and then declined again when terminal differentiation is achieved. HaCaT cells were seeded at different cell densities (post-confluent growth) to induce differentiation. (A) Progressing differentiation was monitored by using an antibody against the differentiation marker involucrin (IVL) in whole cell extracts. 13-Actin served as loading control. (B) Membrane localization of LRRCA8 was visualized by using primary Anti-LRRC8A antibody and FITC-labeled secondary antibody. DNA was counterstained with DAPI to identify the cell nucleus. Note: The green fluorescent FITC signal is shown in white/light grey in this picture.
[0199]
[0200] Western Blot analysis of normally and abnormally differentiating HaCaT cells. HaCaT cells were seeded at different cell densities and cultivated in the presence (B) or absence (A) of pro-inflammatory cytokines (IL-1, IL-17A, TNF-) to mimic psoriatic conditions. Progressing normal (A) and abnormal (B) differentiation was monitored by using antibody against the differentiation marker involucrin (IVL) in whole cell extracts of HaCaT cells. By using Anti-LRRC8A antibody, a bell-shaped expression pattern of LRRC8A was observed during normal differentiation (A), whereas in psoriasis-like HaCaT cells, LRRC8A protein was detected much later and did not decrease at later stages of abnormal differentiation (B). (C) Punch biopsy (6 mm) from diseased, lesional skin (a, d) and non-lesional skin (d, e) of two different psoriasis vulgaris patients (patient 1: a, b; patient 2: d, e) or from healthy donors (c, f) were fixed in 4% PFA and paraffin embedded. 4 m sections were processed routinely. For immunohistochemistry, primary anti-LRRC8A antibody was applied overnight after antigen retrieval with EDTA solution. Histofine Simple Stain AP Multi (Medac Diagnostika, Wedel, Germany) was used for detection, according to the manufacturer's instructions. Nuclei were stained with hematoxylin. Images were acquired by using a Nikon Eclipse slide scanning microscope. Black color indicates antibody binding. Bars represent 100 m. Healthy human skin shows strong LRRC8A staining in the epidermis and especially in the basal layer (c, f). In contrast non-lesional skin of psoriatic patients shows reduced staining for LRRC8A (b, e) while hardly any (d) or very weak (a) staining can be detected in lesional psoriatic skin. Thus, LRRC8A expressions seems to be downregulated during the psoriatic inflammation.
[0201]
[0202] (A) PCR using genomic DNA as template and LRRC8A specific primer pairs results in a 700 bp PCR product in HaCaT wildtype cells (HaCaT-LRRC8A.sup.+/+) and in a 400 bp PCR product in the HaCaT-LRRC8A.sup./ cell clone, confirming the CRISPR-Cas9-induced 300 bp gene deletion of LRRC8A. M, Molecular weight standard. (B) Western blot analysis using whole cell extracts and LRRC8A antibody showed a strong LRRC8A antibody signal at around 100 kDa in HaCaT wildtype cells (HaCaT-LRRC8A.sup.+/+), which is lacking in HaCaT-LRRC8A.sup./ cells, confirming the absence of detectable LRRC8A protein in HaCaT-LRRC8A.sup./ cells. Note the unspecific antibody signal at above 100 kDa, which is present both in wildtype and in knock out cells. -Actin-specific antibody signal was obtained and served as loading control. M, Molecular weight standard. (C) Measuring VRAC activity using hsYFP in HaCaT cells. HaCaT wildtype (WT) and HaCaT LRRC8A.sup./ cells were transduced with adenovirus containing hsYFP gene expression cassette, pre-incubated with isotonic buffer and then treated with iodide-containing isotonic or hypotonic buffer. hsYFP fluorescence was measured and plotted over time. Addition of isotonic buffer did not result in considerable quenching of hsYFP fluorescence whereas hypotonic iodide-containing buffer led to a fast I.sup. influx and strong I.sup.-dependent hsYFP quenching. The speed of I.sup. influx (F/time) after hypotonic stimulation was quantified and used as a measure for VRAC activity. Exemplary raw data (left graph) and determined VRAC activity (depicted as mean value and standard deviation, bar diagram) are shown. Clearly, in HaCaT-LRRC8A.sup./ cells VRAC activity after hypotonic stimulation was almost completely diminished. *** highly statistically significant, student's t-test, p<0.001. (D) Measuring cell volume changes using the volume-sensitive dye calcein in HaCaT cells. HaCaT wildtype (WT) and HaCaT LRRC8A.sup./ cells were loaded with calcein, pre-incubated with isotonic buffer and stimulated with isotonic or hypotonic buffer. Calcein fluorescence was measured and plotted over time. Hypotonic stimulation of HaCaT cells led to a fast increase and subsequent decrease of calcein fluorescence, indicating cell swelling followed by compensatory RVD. The change of calcein fluorescence (F=F.sub.maxF.sub.0) was normalized to baseline fluorescence (F/F.sub.0) and was used as a measure for relative volume increase (cell swelling). The speed of calcein fluorescence decrease (F/time) was used as a measure of relative volume decrease (RVD). Exemplary raw data (left graph) and calculated relative volume increase (top right) and decrease (bottom right) are shown as mean value and standard deviation. Cell swelling was only mildly affected whereas regulatory volume decrease (RVD) was drastically reduced in HaCaT-LRRC8A.sup./ cells compared to HaCaT wildtype cells. *** highly statistically significant, student's t-est, p<0.001.
[0203]
[0204] (A) Western Blot analysis of differentiating HaCaT wildtype cells (HaCaT WT) and HaCaT cells devoid of LRRC8A (HaCaT LRRC8A.sup./). HaCaT cells were seeded at different cell densities (post-confluent growth) to induce differentiation. Progression of differentiation was monitored by using antibodies against the differentiation markers involucrin (IVL) and keratin 10 (KRT10) in whole cell extracts. In cells lacking LRRC8A activity (HaCaT LRRC8A.sup./), IVL occurred in earlier stages of the differentiation process and KRT10 protein level did not continuously increase but stayed at a low level throughout the entire differentiation. (B, C) Pie charts to compare differentially regulated genes of the RNA sequencing data of HaCaT LRRC8A.sup./ cells with published transcriptome studies that are related to psoriasis. Among the 23 genes, which were reported by Chiricozzi et al..sup.5 to be differentially expressed in keratinocytes treated with IL-17, 6 genes were found to be not expressed at all in HaCaT cells, 6 genes not to be affected and almost 50% of the genes (i.e. 11 of the 23 genes, namely: IL1F9, PI3, LCN2, 1L8, S100A8, S100A9, SPRR2D, IL-1B, ALDH1A3, CXCL1, SAT1) to be either up- or downregulated in HaCaT-LRRC8A.sup./ cells (B). Among the 35 genes, which were reported by Swindell et al..sup.7 to be most strongly elevated in psoriasis lesions of patients, it was found by RNA sequencing that 14 genes were not expressed at all in HaCaT cells, 10 genes were not affected, whereas approx. 30% of the genes (i.e. 11 of the 35 genes, namely: TCN1, S100A7A, AKR1B10, PI3, S100A9, 1L36G, LCN2, OASL, IGFL1, KRT16, CXCL1) were also up- or downregulated in HaCaT-LRRC8A.sup./ cells (C).
[0205]
[0206] (A) In healthy skin, keratinocytes develop gradually from proliferating basal cells into spinous, granular and corneal layers of the epidermis. This process is called terminal differentiation and requires a tight balance between proliferation and differentiation. At the beginning proliferation is high in basal cells, whereas differentiation is low (grey triangle). With proceeding terminal differentiation (black arrow), proliferation decreases (dashed triangle) whereas differentiation increases (grey triangle). Normal differentiation also requires dynamic change of LRRC8A expression. The level of LRRC8A (black triangle) first increases, then reaches its maximum and drops again at later stages of normal differentiation. Triangles indicate die increase/decrease of LRRC8A protein levels (black), differentiation (grey) and proliferation (dashed) during the process of terminal differentiation of keratinocytes (black arrow).
[0207] (B) In the skin disorder psoriasis, the balance between differentiation and proliferation is disturbed. Differentiation is slower (grey triangle), whereas proliferation is faster (hyperproliferation, dashed triangle). In this abnormal proliferation process, also LRRC8A expression is disturbed. LRRC8A levels (black triangle) are lower at early stages, increase much slower and do not decline again but instead stay higher in later stages compared to healthy skin.
[0208] (C) When LRRC8A is completely absent, e.g. in case of LRRC8A knock-out cells, or when LRRC8A levels are too low (black line) at early stages, e.g. in case of psoriasis, keratinocytes are not able to undergo their normal differentiation program (grey triangle). In summary, LRRC8A is required for normal epidermal differentiation. In contrast, in psoriasis LRRC8A levels are too low leading to abnormal differentiation. As a consequence, increasing LRRC8A expression or enhancing LRRC8A activity represents a novel approach for treatment of skin conditions that are related to disturbed differentiation, e.g. psoriasis.
[0209]
[0210] The effect of different LRRC8A levels/ion channel activity (black triangle) on differentiation (grey triangle) and proliferation (dashed triangle) and its impact on psoriasis and wound healing are illustrated. Degree of change of psoriasis and wound healing is indicated by a black/grey color gradient, whereby grey indicates improvement and black indicates worsening.
[0211] (A) When LRRC8A activity/level is low (or even completely absent e.g. in case of knock-out cells), it causes aberrant differentiation and hyperproliferation (B), which are characteristics of both psoriasis and wound healing (C). To improve wound healing it is beneficial to further promote this type of epidermal change, meaning that proliferation should be even further increased and differentiation even more reduced (C+D). For treatment of psoriasis the opposite effect is beneficial, meaning that proliferation should be reduced and differentiation enhanced (C+D). This can be achieved by modulating LRRC8A activity: Inhibition of LRRC8A (E) will lead to less differentiation but more proliferation (B), thereby improving wound healing. In contrast, activation of LRRC8A (E) will lead to more differentiation and less proliferation (B), thereby improving psoriasis and similar disorders.
[0212] The following examples illustrate the invention:
Example 1: Material and Methods
[0213] Cell Culture Conditions and Cell Cultivation
[0214] HaCaT cells were cultivated in Dulbecco's Modified Eagle Medium high glucose 4.5 g/L (PAA Laboratories) supplemented with 8% fetal calf serum (Biochrom) and 3.5 mM L-glutamine (PAA Laboratories) at 37 C. and 5% CO.sub.2.
[0215] Normal human epidermal keratinocytes (NHK) are primary keratinocytes which were purchased from PromoCell or were isolated from human juvenile foreskin (according to 50). Cells were cultured in keratinocyte growth medium 2 (PromoCell) supplemented with keratinocyte growth medium 2 supplement Mix (PromoCell) containing 0.004 ml/ml bovine pituitary extract, 0.125 ng/ml epidermal growth factor, 5 g/ml insulin, 0.33 g/ml hydrocortisone, 0.39 g/ml epinephrine, 10 g/ml transferrin and 0.06 mM CaCl.sub.2 up to a confluence of 80%.
[0216] Gene Expression Analysis Using RT-PCR
[0217] To determine whether and which LRRC8 family members (LRRC8A-E) are expressed in the keratinocyte cell line HaCaT and in normal human epidermal keratinocytes (NHK), RT-PCR was performed. For this purpose, total RNA from HaCaT and NHK cells was extracted by using NucleoSpin RNA II Kit (Macherey-Nagel). cDNA was synthesized by reverse transcription using 1 g total RNA and Poly-dT primer (ProtoScript M-MuLV First Strand Synthesis Kit, New England Biolabs). PCR amplification of LRRC8A-E was performed using gene specific oligonucleotide primers and Phire Hot Start II PCR Master Mix (Thermo Scientific) including initial denaturation at 98 C. for 30 sec followed by 27 amplification cycles comprising denaturation at 98 C. for 5 sec, primer annealing at 55 C. for 5 sec, and elongation at 72 C. for 12 sec followed by a final elongation step at 72 C. for 1 min. PCR amplification products were separated on 2% agarose gels and visualized by Midori Green (Biozym) staining. The following sense and antisense oligonucleotides were used:
TABLE-US-00003 -Actinfw (SEQIDNO:42) 5-GTGGGGCGCCCCAGGCACCA-3; -Actinrv (SEQIDNO:43) 5-CTCCTTAATGTCACGCACGATTTC-3; LRRC8Afw (SEQIDNO:44) 5-CCTGCCTTGTAAGTGGGTCAC-3; LRRC8Arv (SEQIDNO:45) 5-CACAGCGTCCACGTAGTTGTA-3; LRRC8Bfw (SEQIDNO:46) 5-CTGGCATAGAAAGCCCAACTT-3; LRRC8Brv (SEQIDNO:47) 5-CGATTTCAAGAGTGATGTGGGT-3; LRRC8Cfw (SEQIDNO:48) 5-CTGGGGAAGTGTTTTGACTCTC-3; LRRC8Crv (SEQIDNO:49) 5-GGACCAGATTGGATGGTGTTG-3; LRRC8Dfw (SEQIDNO:50) 5-GTGGTCTGTTTGCCAGTATTGC-3; LRRC8Drv (SEQIDNO:51) 5-CCCAAAGGAAATGTCGTTTGTTG-3; LRRC8Efw (SEQIDNO:52) 5-CAAGCAGTTCACGGAACAGC-3; LRRC8Erv (SEQIDNO:53) 5-GGGCCTCTGATAAGTTCTCCTG-3.
[0218] Protein Detection Using Western Blot Analysis
[0219] Total protein was extracted by incubation of cells with RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% Deoxycholate, 0.1% SDS, 1 mM EDTA) for 30 min at 4 C. Supernatant was centrifuged for 1 min and protein extracts were denatured by incubation with Laemmli sample buffer (0.5 M Tris pH 6.8, 4% SDS, 40% glycerol, 100 mM DTT, 0.08% bromophenol blue) for 10 min at 95 C. Protein concentration was determined by BCA assay (Thermo Fisher). 10-15 g total protein was separated on 7.5% or 12.5% SDS-PAA gels and analysed by western blot using specific antibodies against LRRC8A (Novus NBP2-32158 1:500), Cytokeratin 10 (abcam ab76318, 1:10000), Involucrin (abcam ab20202, 1:10000), -Actin (Sigma Aldrich A1978, 1:10000) and -Tubulin (Sigma Aldrich T9026, 1:5000) and species specific secondary antibodies (a-mouse IgG, a-rabbit IgG, VWR, 1:5000). Protein was visualized by ECL reagent (Merck Chemicals) and sizes of proteins were estimated by comparison with protein marker IV (10-170 kDa) (PeqLab).
[0220] Immunohistology and Immunofluorescence Staining of Skin Specimen
[0221] Healthy individuals were recruited and gave written informed consent. The study was approved by the ethics committee of the Clinic of the Goethe-University (116/11); the Declaration of Helsinki protocols were followed. Punch biopsies (6 mm) were taken. For immunohistochemistry they were fixed in 4% PFA, paraffin embedded and cut into 4 m sections. Paraffin sections were processed routinely.sup.51. Primary antibody (anti-Swell Novus 1:100; also available under the name anti-LRRC8A NBP2-32158 from Novus Biologicals)) or rabbit isotype control antibody (#3900 Cell Signaling, 1:2,5) was applied overnight after antigen retrieval with citrate solution pH 6. Histofine Simple Stain AP Multi (Medac Diagnostika) was used for detection, according to the manufacturer's instructions. Images were acquired by using a Nikon Eclipse slide scanning microscope. For immunofluorescence staining biopsies were collected in TissueTek OCT (Sakura) and cut into 8 m cryosections, fixed in methanol. Specimens were blocked with 5% goat serum/TBS-T and incubated overnight at 4 C. with primary anti-Swell1 Novus 1:100 or isotype antibody (#3900 Cell Signaling, 1:2,5). After washing with TBS, samples were incubated with AlexaFluor488 labeled secondary antibody (1:1000 LifeTechnologies) and nuclei were stained with DAPI. Confocal images were generated using a ZeissLSM510 microscope.sup.4.
[0222] Immunofluorescence Staining of Cells
[0223] HaCaT cells were seeded on glass slides, fixed in methanol and permeabilized with TBS-T. Specimens were blocked with 5% goat serum/TBS-T and incubated overnight at 4 C. with primary or isotype antibodies. After washing, samples were incubated with AlexaFluor488 labeled secondary antibody and nuclei were stained with DAPI. Confocal images were generated using a ZeissLSM510 microscope.sup.52.
[0224] Differentiation of Keratinocytes In Vitro
[0225] Differentiation of HaCaT cells was initiated by post-confluence growth. Therefore HaCaT cells were cultivated in increasing cell numbers (0.1 up to 1.0*10.sup.6 cells/6 well) for 48 h resulting in sub-confluent to post-confluent cell density as described in Buerger C. et al..sup.4. Then, cells were harvested and used for RNA extraction, protein extraction or were fixed for immunofluorescence analysis. If indicated, cells were treated with a mix of inflammatory cytokines, consisting of IL-1b, IL-17A and TNF- (from Peprotech; 20 ng/ml each) for the indicated period. Then, cells were harvested and used for RNA extraction, protein extraction.
[0226] Generation of LRRC8A Knock-Out Cell Lines by CRISPR/Cas9
[0227] To generate monoclonal HaCaT cells devoid of the LRRC8A gene, HaCaT cells were transduced with adenovirus Ad5-CMV-Cas9-wt-2A-OFP (produced by Sirion Biotech) delivering a gene expression cassette encoding for Cas9 nuclease and orange fluorescent protein under the control of the CMV promoter and with adenovirus Ad5-U6-sgRNA-G-SWELL1-U6-sgRNA-G-SWELL4 (produced by Sirion Biotech) delivering a gene expression cassette encoding two single guide RNAs targeting different positions of LRRC8A under the control of the U6 promoter. The sequence of used sgRNAs are as followed:
TABLE-US-00004 sgRNA-G-LRRC8A#1: (SEQIDNO:54) 5-GCTGCGTGTCCGCAAAGTAG; and sgRNA-G-LRRC8A#4: (SEQIDNO:55) 5-CCGGCACCAGTACAACTACG.
[0228] After adenoviral transduction, monoclonal HaCaT-LRRC8A.sup./ cells were isolated from the heterogeneous cell pool by limiting dilution. The Cas9-mediated genomic deletion of LRRC8A was then confirmed by target site-specific PCR and subsequent Sanger sequencing. PCR amplification of the genomic region was performed using specific oligonucleotides (Seq_LRRC8A_24839 fw 5-TGGTTTCCCAGCCAAGTG (SEQ ID NO:56); and Seq_LRRC8A_965 rv 5-GCGGGAATTTGAACCAGAAG (SEQ ID NO:57)), dNTPs, Phusion Polymerase (Thermo Fischer) and genomic DNA as template. Genomic DNA was initially denatured at 98 C. for 30 sec followed by 30 amplification cycles including 10 sec denaturation at 98 C., 10 sec annealing at 65 C. and 30 sec elongation at 72 C. and a final elongation of 10 min at 72 C. PCR amplification products were separated on 2% agarose gels and visualized by Midori Green (Biozym) staining. PCR amplification products were purified using QuiQuick PCR Purifictaion Kit (Quiagen) and sequenced by Sanger sequencing using oligonucleotide Seq_LRRC8A_24839 fw (SEQ ID NO:56).
[0229] Measuring of Swelling-Induced VRAC Activity Using hsYFP
[0230] To measure VRAC activity in tissue culture cells the hsYFP gene expression cassette is delivered via adenoviral transduction. Therefore 0.2510.sup.6 cells/well HaCaT cells were seeded in 6-well plates and incubated at 37 C. and 5% CO.sub.2 for 1 day. Then, cells were transduced with adenovirus Ad-CMV-hsYFP (Sirion) at 300 MOI (multiplicities of infection) and incubated at 37 C. and 5% CO.sub.2 for an additional day. 30,000 transduced cells were seeded in 96-well black-walled, clear bottom microplates (Costar) and cultivated for 1 day. Cells were washed three times with 70 l isotonic incubation buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM glucose, 10 mM HEPES/NaOH, pH7.2, 329 mOsm) and incubated with 50 l isotonic incubation buffer for 15 min at 37 C. Cellular hsYFP fluorescence (excitation at 485 nm, emission at 535 nm) was continuously recorded every 3.5 sec in an automated fluorescence plate reader. After baseline recording for 20 sec, cells were stimulated by addition of isotonic I.sup.-solution (70 mM Nal, 5 mM NaCl, 140 mM mannitol, 5 mM KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM glucose, 10 mM HEPES/NaOH, pH7.2, 329 mOsm) or hypotonic I.sup.-solution (70 mM Nal, 5 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM glucose, 10 mM HEPES/NaOH, pH7.2, 189 mOsm) to establish an extracellular I.sup. concentration of 50 mM I.sup.. Addition of hypotonic buffer resulted in a 30% decrease in osmolarity (final osmolarity of 229 mOsm). For experiments without extracellular calcium, 2 mM CaCl.sub.2 were replaced by 2 mM MgCl.sub.2.
[0231] HsYFP fluorescence (arbitrary units) was plotted over time and I.sup. influx rate was derived from the initial slope (F fluorescence/time) as a measure of swelling induced ion channel activity. VRAC activity was determined from at least four independent experiments performed in duplicates and were presented as the meanstandard deviation (SD). A two-sided, unpaired Student's t-test was performed for statistical analysis and significance was indicated as * p<0.05, **p<0.01 and ***p<0.001.
[0232] Measuring of Cell Volume Changes Using Calcein-AM
[0233] 30,000 cells were seeded in 96-well black-walled, clear bottom microplates (Costar) and cultivated for 1 day. Prior to measurement, cells were loaded with 10 M Calcein-AM (Fisher Scientific) in normal cultivation medium for 75 min at 37 C. For experiments investigating the influence of intracellular calcium, cells were additionally loaded with 20 M BAPTA-AM. Cells were washed three times with 70 l isotonic incubation buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM glucose, 10 mM HEPES/NaOH, pH7.2, 329 mOsm) and incubated in 50 l isotonic incubation buffer at 37 C. Cellular calcein fluorescence (excitation at 485 nm, emission at 538 nm) was continuously recorded every 1.6 sec in an automated fluorescence plate reader. After baseline recording for 20 sec, cells were stimulated by addition of isotonic solution (20 mM NaCl, 250 mM mannitol, 5 mM KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM glucose, 10 mM HEPES/NaOH, pH7.2, 329 mOsm) or hypotonic solution (20 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM glucose, 10 mM HEPES/NaOH, pH7.2, 79 mOsm). Addition of hypotonic buffer resulted in a 55% decrease in osmolarity (final osmolarity of 150 mOsm). Calcein fluorescence (arbitrary units) was plotted over time. Fluorescence (F=F.sub.maxF.sub.0) before (F.sub.0) and after (F.sub.max) stimulation was normalized to baseline fluorescence (F/F.sub.0) and used as a relative measure for cell volume increase. Fluorescence quenching was derived from the slope (F fluorescence/time) after maximal fluorescence increase (F.sub.max) and used as a measure of cell volume decrease. Cell volume changes were measured in two independent experiments carried out in triplicates and was presented as the meanSD. A two-sided, unpaired Student's t-test was performed for statistical analysis and significance was indicated as * p<0.05, **p<0.01 and ***p<0.001.
[0234] Transcriptome Data Analysis of HaCaT Wildtype and LRRC8A Knock-Out Cells
[0235] Cell lysis and RNA isolation were performed using the NucleoSpin RNA Kit II from Macherey-Nagel. For library preparation, the TruSeq RNA Library Prep Kit v2 from Illumina was used starting with an input amount of 500 ng of total RNA. The prepared libraries were sequenced with a 2150 bp read length using the HiSeq 3000/4000 SBS Kit and an Illumina Hiseq 4000 sequencer. The adapter trimmed, demultiplexed and quality filtered reads were aligned to the hg19 reference genome and transcriptome using Hisat2 (2.0.4).sup.53. The Hisat2 output files (SAM) were converted to the BAM format and were sorted and indexed using SAMtools (1.3.1).sup.54. The BAM files were evaluated by Cuffdiff2 (2.1.1), as previously described.sup.55. The differential transcript abundance was calculated using Cuffdiff2. All samples were compared and evaluated in one calculation cycle, allowing the algorithm to estimate the FPKM values at the transcript level resolution and to control for variability across the replicate libraries.sup.55. Cuffdiff2 was used because its normalization of read counts is equal to that of DESeq.sup.55, which has been shown to be one of the most reliable normalization methods for RNA sequencing.sup.56. Here, a scaling factor for each gene in a given sample was calculated as the median of the ratio of the read count of that gene over its geometric mean across all samples.sup.57.
Example 2: LRRC8A is Expressed in Cultured Human Keratinocytes and in Human Skin
[0236] First it was asked whether LRRC8A and its other LRRC family members (LRRC8B-E) are expressed in human skin cells. RT-PCR analysis was performed and showed that mRNA transcripts for all LRRC8 subunits are readily detectable in cells of the keratinocyte cell line HaCaT as well as in primary normal human epidermal keratinocytes (NHKs) (
[0237] It was next analyzed whether LRRC8A is also found in the context of normal human skin. LRRC8A-antibody staining was indeed observed in histological sections of human skin biopsies (
Example 3: LRRC8A is Dynamically Regulated During Keratinocyte Differentiation
[0238] Since the gradual distribution of LRRC8A proteins along the epidermal keratinocyte layers (
Example 4: LRRC8A Expression is Changed in Inflammatory, Psoriasis-Like Keratinocytes and in Psoriasis Skin Lesions
[0239] After having found that LRRC8A is dynamically regulated during normal differentiation (
[0240] Next it was asked whether LRRC8A is also changed in primary psoriatic keratinocytes and in skin lesions of psoriasis patients. Since it is difficult to obtain and analyze psoriatic skin biopsy samples, a search in publicly available whole transcriptome data from psoriatic skin lesions was initially conducted. Several studies were identified which gathered transcriptome data from primary keratinocytes treated with TNF- and IL-17, which mimics a psoriatic, inflammatory skin cell, as well as studies from psoriasis patients.sup.5,7,41-43. Evaluation of the data yielded that these studies focused on the importance of the most strongly affected genes in psoriasis. These are approx. 35 genes, which can be considered as key deregulated genes in psoriasis.sup.7, albeit, more than 200 genes.sup.5,41 and up to 2200 genes.sup.7 were described to be additionally changed. The function of many of these genes is not known and, therefore, scientific studies at present primarily address the function of the known genes. As a consequence, new potential key players and targets in psoriasis are often dismissed or overseen.
[0241] Nonetheless, the available transcriptome data were searched and it was asked whether LRRC8A is one of the so far overseen deregulated genes in psoriasis. Indeed, analysis of the supplementary data revealed that LRRC8A belongs to the list of differentially regulated genes in primary keratinocytes treated with TNF- and IL-17.sup.5 (Table 3), which is in perfect accordance with the results from the psoriasis-like HaCaT cell model provided herein (
TABLE-US-00005 TABLE 3 The psoriatic model investigated, log2 change and fold change of gene expression based on transcriptome data are listed. Two genes of the LRRC8 gene family, LRRC8A and LRRCB, are upregulated by approx. 1.5-2 in keratinocytes treated with TNF- and/or IL-17, which mimics a psoriatic inflammatory skin cell, or in pathogenic keratinocytes of psoriasis patients. log2 fold Gene Study Psoriatic model change change LRRC8A Chiricozzi et al. TNF- 0.510 1.42 LRRC8B Chiricozzi et al. IL-17 + TNF- 0.562 1.47 LRRC8B Suarez-Farinas et al. Patients 1.100 2.14
[0242] Next it was investigated whether LRRC8A expression is changed in human skin of psoriasis patients (
Example 5: Reduction of LRRC8A Activity by CRISPR/Cas9 Approach Influences VRAC Activity and RVD in HaCaT Keratinocytes
[0243] The deregulated expression of LRRC8A in psoriasis renders it a potentially attractive new target to modulate differentiation in psoriasis or other skin disorders such as atopic dermatitis, with the aim to attenuate the differentiation defects in the diseased skin cells. In order to prove that modulation of LRRC8A activity can be employed to manipulate the differentiation process, the differentiation process in the absence of LRRC8A activity was monitored.
[0244] For this purpose, a HaCaT-LRRC8A.sup./ knock out cell line, which is devoid of functional LRRC8A, was created by employing the CRISPR-Cas genome editing technology. Two single-guide RNAs (sgRNA-G-LRRC8A #1: 5-GCTGCGTGTCCGCAAAGTAG (SEQ ID NO:54); sgRNA-G-LRRC8A #4: 5-CCGGCACCAGTACAACTACG (SEQ ID NO:55)) were designed to tether the Cas9 nuclease to defined regions in the genomic LRRC8A loci, which then leads to the defined deletion of approx. 300 bp of the LRRC8A coding sequence. The predicted LRRC8A gene deletion was confirmed by PCR using specific oligonucleotide primer pairs (Seq_LRRC8A_24839 fw 5-TGGTTTCCCAGCCAAGTG (SEQ ID NO:56); Seq_LRRC8A_965 rv 5-GCGGGAATTTGAACCAGAAG (SEQ ID NO:57)) and isolated genomic DNA (
[0245] It was then analyzed whether these HaCaT cells devoid of LRRC8A have a reduced VRAC activity and whether this influences cell volume regulation. Cell volume changes were determined by loading HaCaT cells with the fluorescent volume-sensitive dye calcein.sup.18,58,59, whereas VRAC activity was determined by expressing the fluorescent halide-sensitive YFP (hsYFP), which allows to monitor the characteristic I.sup. influx of chloride channels including VRACs.sup.18,31,60-62.
[0246] Strikingly, HaCaT-LRRC8A.sup./ cells completely lacked VRAC activity upon hypotonic stimulation (
Example 6: Modulation of LRRC8A Activity Influences Differentiation of Keratinocytes
[0247] After having established that the LRRC8A activity is almost completely diminished in HaCaT-LRRC8A/ cells, this cell line was used to monitor the effect on keratinocyte differentiation in the absence of LRRC8A activity (
[0248] To obtain a more comprehensive overview of other potentially deregulated genes, RNA-Seq analysis of HaCaT-LRRC8A.sup./ cells was performed. In addition to IVL and KRT10 this transcriptomics approach revealed differential gene expression of additional crucial keratinocyte differentiation markers such as TGM1, KRT4 and KRT15 and markers for abnormal hyperproliferation KRT6 and KRT16. It was also found that several key genes, which were described to be deregulated in psoriasis.sup.5,7 are also deregulated in HaCaT cells devoid of LRRC8A (HaCaT-LRRC8A.sup./ cells) (
[0249] In particular, Chiricozzi et al. had focused on a common set of 23 genes that are differentially expressed in keratinocytes treated with IL-17.sup.5. It was found by RNA sequencing in the present study that among those 23 genes, 6 genes were not expressed at all in HaCaT cells, 6 genes were not affected, and almost 50% of the genes (i.e. 11 of the 23 genes) were also deregulated in HaCaT-LRRC8A.sup./ cells (
[0250] In the second study, Swindell et al. reported a group of 35 genes that are most strongly elevated in psoriasis lesions of patients.sup.7. Among those 35 genes, 14 genes were found by RNA sequencing that are not expressed at all in HaCaT cells, 10 genes were not affected, and approx. 30% of the genes (i.e. 11 of the 35 genes) were also deregulated in HaCaT-LRRC8A.sup./ cells (
[0251] In summary, it was shown that a decrease in LRRC8A expression/activity leads to changed differentiation, i.e. it causes aberrant differentiation. Consequently, increasing LRRC8A activity will have beneficial effects on abnormal differentiation process, such as those found in psoriasis. Thus, differentiation can be affected by modulating LRRC8A activity, which can be used for the therapeutic treatment of differentiation defects, for example in psoriasis, or for cosmetically alleviating the effects of a skin condition, such as psoriasis, on the appearance of the skin of an affected individual.
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