QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 29 July 2008
doi: 10.1242/jcs.025643

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ojeh, N. Articles by Määttä, A. Search for Related Content PubMed PubMed Citation Articles by Ojeh, N. Articles by Määttä, A. Social Bookmarking                         What's this?

The MAGUK-family protein CASK is targeted to nuclei of the basal epidermis and controls keratinocyte proliferation

School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK

View larger version (115K): [in this window] [in a new window]   Fig. 1. Localisation of CASK expression in skin epidermis and developing hair follicles. (A,B) Immunofluorescence staining of the skin of newborn mice using a rabbit polyclonal anti-CASK antibody (A, B, green channel) and monoclonal JoL-4 antibody against lamin-A (red channel, AI) or a monoclonal Ki67 antibody (red channel, BI). Note the predominant nuclear staining for CASK in the basal layer of epidermis. Arrows indicate colocalisation of CASK and lamin A (AII) and CASK and Ki67 (BII); dashed lines indicate basement-membrane zone. SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale; HF, hair follicle. (C,D) Immunofluorescence staining of CASK in (C) adult mouse skin and (D) human skin. Dashed lines indicate basement-membrane zone. (E) CASK expression in mouse primary keratinocytes within nuclei, cytoplasm and at cell borders (arrowheads). (F) Immunoblotting of whole-cell protein (W), nuclear (N) and cytosolic (C) extracts from mouse primary keratinocytes probed with antibodies against CASK and -tubulin. (G-K) Strong nuclear CASK expression is seen during rat hair-follicle development. Skin from whisker pads of E18 rats was immunostained for CASK (green) and the adherens-junction marker protein β-catenin (red). (G) Epithelial placode. (H,I) Hair germs. (J) Hair peg. (K) Developing follicle. (L) Western blot analysis of epidermal protein extracts from wild-type (WT), K14-human syndecan-1-transgenic (Tg) and syndecan-1 null (KO) mice probed with antibodies against CASK and involucrin. (M) CASK staining of skin from WT, Tg and KO mice. Notice the increased expression in the skin of Tg mice. Scale bars, 50 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 2. CASK localisation is altered during keratinocyte differentiation. (A) HaCaT cells were grown in low-Ca2+ (0.06 mM) DMEM or in high-Ca2+ (1.6 mM) DMEM for 1 day (D1) and double stained with CASK (green channel) and a monoclonal antibody against E-cadherin (red channel) as indicated. (B) HaCaT cells were grown in low-Ca2+ or high-Ca2+ DMEM for 6 hours (6H), 1 (D1), 3 (D3) or 5 (D5) days and stained using a rabbit polyclonal antibody against CASK. Notice the gradual disappearance of nuclear CASK in cells grown in high-Ca2+ DMEM over time. (C) Nuclear (N) and cytosolic (C) extracts from HaCaT cells grown under low or high-Ca2+ conditions for 1 day (D1) and 5 days (D5), and immunoblotted using antibodies against CASK and -tubulin. (D) Human primary keratinocytes were grown in low-Ca2+ defined keratinocyte serum-free (SFM) medium or in high-Ca2+ SFM medium for 1 day (D1) or 3 days (D3) and stained with rabbit polyclonal antibody against CASK (green channel) and a monoclonal involucrin antibody (red channel). Scale bars, 50 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 3. CASK expression in HaCaT keratinocytes upon cell-cycle re-entry. (A) Serum-starved HaCaT cells were re-stimulated to enter the cell cycle by adding serum for 0, 6, 12, 22 or 30 hours, and cell-cycle analysis was performed at each time point to verify the stage of the cell cycle (graphs on right). Cells were co-stained with a rabbit polyclonal anti-CASK antibody (green channel) and a mouse monoclonal anti-Ki67 antibody (red channel) at each time point. Mitotic cells were seen at 30 hours (arrowheads). (B) Cells were harvested at the indicated times after each serum stimulation and immunoblotting was performed using antibodies against CASK (112 kDa) and -tubulin (50 kDa). (C) Expression of cyclin A (60 kDa) and cyclin B (62 kDa) at indicated times after re-stimulation. Scale bars, 50 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 4. CASK expression in HaCaT cells following release from cell cycle blockers. (A,B) Serum-starved HaCaT cells were re-stimulated to enter the cell cycle by adding serum. (A) Cells were treated with 10 µM BRDU for 45 minutes prior to staining with rabbit polyclonal anti-CASK antibody (green channel) and mouse monoclonal anti-BrdU antibody (red channel) at 12 and 22 hours. (B) Cells were co-stained with anti-CASK antibody (ZYMED; green channel) and mouse monoclonal anti-H3-P antibody for mitotic cells (red channel) at 22 and 30 hours. (C) Cells synchronised with Aphidicolin were stained with a rabbit polyclonal anti-CASK antibody (ZYMED; green channel) and mouse monoclonal anti-PCNA antibody (red channel). Corresponding cell-cycle analysis is shown in the graph at right. (D) Cells were synchronised with nocodazole prior to staining with a rabbit polyclonal anti-CASK antibody (ZYMED; green channel) and a mouse monoclonal anti-H3 antibody (red channel). Corresponding cell cycle analysis is shown at right. Scale bars, 50 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 5. Biochemical fractionation and in situ nuclear-matrix extraction of HaCaT cells. (A) Western blotting. HaCaT cells grown in low-Ca2+ medium were subjected to a sequential extraction by CSK 0.1% Triton X-100 (C/T, 0.1%), CSK 0.5% Triton X-100 (C/T, 0.5%), chromatin digestion by DNase I (DNAse) and a final extraction by 0.25 M ammonium sulfate. Whole-cell protein extracts (P1) were prepared and, following each step, insoluble fractions P2, P3, P4 and P5 were pelleted, and soluble fractions S2, S3, S4 and S5 were retained for immunoblotting with monoclonal anti-CASK antibody and JoL2 antibody to detect lamin A/C. (B) HaCaT cells were grown on glass coverslips in low-Ca2+ medium and then subjected to in situ nuclear-matrix extraction using five sequential treatments with CSK buffer only (no extraction; stage I), CSK 0.1% Triton X-100 (stage II), CSK 0.5% Triton X-100 (stage III), DNase I digestion (stage IV) and 0.25 M ammonium sulfate extraction (stage V), and processed for indirect immunofluorescence microscopy using antibodies against CASK (green channel) and lamin A/C (red channel). (C,D) HaCaT cells grown in low-Ca2+ DMEM were transfected with a full-length CASK-GFP construct and subjected to in situ nuclear-matrix extraction using sequential treatments up to the indicated stages. GFP-CASK is shown in the green channel and lamin A/C in the red channel (C). Stages IV and V extracted cell showing colocalisation (arrowheads) of the nucleolus marker, nucleolin (red channel) with GFP-CASK (green channel) (D). Scale bars, 50 µm (B), 20 µm (C,D).

View larger version (38K): [in this window] [in a new window]   Fig. 6. RNAi of CASK leads to increased cell proliferation in HaCaT keratinocyte monolayer cultures. (A) Efficiency of CASK RNAi. HaCaT cells were transfected with control siRNA or three different siRNAs targeting CASK for 96 hours, and then harvested for immunoblotting and probed with antibodies against CASK and -tubulin. (B) Growth of non-synchronised HaCaT cells transfected with control siRNA, CASK siRNA1 or CASK siRNA2. Cells were subjected to the MTT proliferation assay at 72 hours post siRNA transfection. Error bars indicate +s.d. (n=3). (C) Growth curves of control-siRNA- and CASK-siRNA-transfected HaCaT cells. Serum-starved and re-stimulated cells were transfected with control siRNA or CASK siRNA, and cells were subjected to MTT proliferation assay at 0, 24, 48, 72 and 96 hours post siRNA transfection. Error bars indicate ±s.d., (n=3). (D) Phase-contrast micrographs of control-siRNA and CASK-siRNA-transfected HaCaT cells at 24, 48, 72 or 96 hours post siRNA transfection. Scale bars, 100 µm. (E) Protein extracts from control-siRNA and CASK-siRNA-transfected HaCaT cells were immunoblotting and probed with antibodies against CASK, phosphorylated Rb (pRb), total Rb and actin. (F) FACS cell-cycle analysis of DNA content of control-siRNA and CASK-siRNA-transfected synchronised HaCaT cells at 0 and 60 hours post siRNA transfection. Percentages of cells in G1 and S phases, and at G2 or M phase are indicated. (G) FACS cell-cycle analysis of DNA content of control-siRNA and CASK-siRNA-transfected non-synchronised HaCaT cells at 60 hours post siRNA transfection. Percentages in G1 and S phases, and at G2 or M phase are indicated.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Proliferation, growth factor responses, apoptosis and basal integrin expression in siRNA-transfected synchronised HaCaT cells at 60 hours post siRNA transfection. (A) Cell-cycle analysis of DNA content in cells pulse labelled with EdU. Percentages of cells in G1 and S phases, and at G2 or M phase are indicated. (B) Control-siRNA- and CASK-siRNA-transfected cells were serum starved and left untreated, or were treated with 10 ng/ml KGF or 10 ng/ml TGF for 24 hours. Proliferation was substantially enhanced in CASK-siRNA-transfected cells treated with KGF or TGF for 24 hours compared with control-siRNA-transfected cells treated with the same growth factors. (C) Percentage of apoptotic cells. Cells were harvested and subjected to staining for apoptosis using an apoptosis detection kit (BD Biosciences). Positive-control cells provided in the kit show a high percentage of apoptosis compared with negligible levels seen in either the control or CASK siRNA transfected cells. (D) Basal integrin expression. Cells were harvested, stained for 6, β1 or β4 integrin and subjected to analysis by flow cytometry.

View larger version (95K): [in this window] [in a new window]   Fig. 8. Knockdown of CASK leads to epidermal hyperproliferation in organotypic cultures. (A) Protein extracts from epidermal tissue were isolated from organotypic cultures, and prepared with control-siRNA- and CASK-siRNA-transfected HaCaT cells after 7 days at the air-liquid interface. Immunoblotting was performed using antibodies against CASK (112 kDa), PCNA (30 kDa), Myc (64 kDa), involucrin (120 kDa) and filaggrin (40 kDa), actin (42 kDa) was used as an internal control. Notice the reduced CASK protein level in CASK-siRNA-transfected epidermal extracts concomitant with the upregulation of PCNA and Myc. (B) H&E histology and immunofluorescence staining for CASK in control and CASK-knockdown HaCaT raft cultures. H&E histology shows a thicker epidermal morphology in the CASK-knockdown 3D cultures, consisting of several layers compared with the control-siRNA-transfected rafts. (C) Expression of keratins, proliferation and differentiation markers, and basal integrins in siRNA-transfected raft cultures. Notice the presence of Ki67-positive cells and H3-P-positive mitotic cells in the CASK-knockdown skin model, in both basal and suprabasal layers of the epidermis. Involucrin and filaggrin are expressed at similar levels in the suprabasal and differentiating layers of the epidermis. CASK knockdown skin model shows integrin expression also in lateral and apical membranes of the basal cells. All protein staining are shown in green and DAPI nuclear staining is shown in blue. Scale bars, 50 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 9. CASK expression during skin-wound healing. (A) H&E histology of day 3 mouse back-skin full-thickness wound (top panel). Boxed areas that were investigated for CASK expression are shown in magnification (bottom panel). Wound site (left); wound edge (middle); away from wound (right). Frozen sections of wounded skin were immunostained using a rabbit polyclonal anti-CASK antibody. Notice the reduced staining for CASK in keratinocytes at the wound site (WS). (B,C) Knockdown of CASK results in accelerated cell adhesion and spreading. HaCaT cells were transfected with control-siRNA or CASK-siRNA oligonucleotides for 96 hours and seeded onto collagen-coated substrates. After 30 minutes, 1 or 2 hours, attached cells were quantified by an adhesion assay (B) or stained for the focal adhesion marker vinculin (green) (C). DAPI nuclear staining is shown in blue. Scale bars, 50 µm (A), 20 µm (C).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter