Angiopoietin-like 4 regulates epidermal differentiation

Abstract

The nuclear hormone receptor PPARβ/δ is integral to efficient wound re-epithelialization and implicated in epidermal maturation. However, the mechanism underlying the latter process of epidermal differentiation remains unclear. We showed that ligand-activated PPARβ/δ indirectly stimulated keratinocyte differentiation, requiring de novo gene transcription and protein translation. Using organotypic skin cultures constructed from PPARβ/δ- and angiopoietin-like 4 (ANGPTL4)-knockdown human keratinocytes, we showed that the expression of ANGPTL4, a PPARβ/δ target gene, is essential for the receptor mediated epidermal differentiation. The pro-differentiation effect of PPARβ/δ agonist GW501516 was also abolished when keratinocytes were co-treated with PPARβ/δ antagonist GSK0660 and similarly in organotypic skin culture incubated with blocking ANGPTL4 monoclonal antibody targeted against the C-terminal fibrinogen-like domain. Our focused real-time PCR gene expression analysis comparing the skin biopsies from wildtype and ANGPTL4-knockout mice confirmed a consistent down-regulation of numerous genes involved in epidermal differentiation and proliferation in the ANGPTL4-knockout skin. We further showed that the deficiency of ANGPTL4 in human keratinocytes and mice skin have diminished expression of various protein kinase C isotypes and phosphorylated transcriptional factor activator protein-1, which are well-established for their roles in keratinocyte differentiation. Chromatin immunoprecipitation confirmed that ANGPTL4 stimulated the activation and binding of JUNB and c-JUN to the promoter region of human involucrin and transglutaminase type 1 genes, respectively. Taken together, we showed that PPARβ/δ regulates epidermal maturation via ANGPTL4-mediated signalling pathway.

Figure 1. ANGPTL4 deficiency in mice impairs epidermal differentiation.

(A) PPARβ/δ regulates keratinocyte differentiation requires de novo transcription and translation. Relative fold change in mRNA levels of cytokeratin 10, involucrin and transglutaminase type 1 in control (K_CTRL) and PPARβ/δ-knockdown (K_PPARβ/δ) human keratinocytes treated with either DMSO vehicle or PPARβ/δ agonist GW501516 (GW, 100 nM), in the absence or presence of RNA synthesis (actinomycin D, Act-D) or protein synthesis (cycloheximide, CHX) inhibitors as determined by quantitative real-time PCR. Act-D and CHX treatment alone did not affect the transcript level. Ribosomal protein L27 was used as a normalizing reference gene. Values are mean ± SEM of three independent experiments. (B) Haematoxylin and eosin (H&E) and immunofluorescence staining of skin biopsies from wildtype (ANGPTL4^+/+) and ANGPTL4-knockout (ANGPTL4^−/−) mice. Early (cytokeratin 10, CK10), late (fillaggrin, FIL) differentiation markers, proliferating (Ki67) and apoptotic (TUNEL) cells were identified using indicated antibodies or assay. White dotted lines indicated epidermis-dermis junctions. Sections were counterstained with DAPI (blue). Scale bars represent 40 µm. (C) Representative immunoblot of early differentiation (cytokeratin 10, CK10), terminal differentiation (transglutaminase type I, Tgase 1), proliferation (PCNA and cyclin D1), and apoptosis (cleaved caspase 3) markers in ANGPTL4+/+ and ANGPTL4−/− skin biopsies. Immunoblot data are from three independent experiments performed in duplicate. β-tubulin serves as a loading and transfer control.

Figure 2. Organotypic skin coculture (OTC) of ANGPTL4-deficient human primary keratinocytes displayed impaired epidermal differentiation.

(A) Relative mRNA and/or protein levels of ANGPTL4 and ANGPTL3 in human primary keratinocytes transduced with either scrambled control (K_CTRL) or ANGPTL4 siRNA (K_ANGPTL4). Values below immunoblot bands represent the mean fold differences in protein expression level when compared with K_CTRL from 3 independent experiments. (B) Haematoxylin and eosin (H&E) and immunofluorescence staining of OTC sections constructed with either control (K_CTRL) or ANGPTL4-knockdown (K_ANGPTL4) human keratinocytes. Late (fillaggrin, FIL) differentiation markers, proliferating (Ki67) and apoptotic (TUNEL) cells were identified using indicated antibodies or assay. White dotted lines indicated epidermis-dermis junctions. Sections were counterstained with DAPI (blue). Scale bars represent 40 µm. (C) Representative immunoblot of early epidermal differentiation (cytokeratin 10, CK10), terminal differentiation (transglutaminase type I, Tgase 1), proliferation (PCNA and cyclin D1), and apoptosis (cleaved caspase 3) markers in isolated epidermis of indicated OTCs. All immunoblot data are from three independent experiments performed in duplicate. β-tubulin serves as a loading and transfer control.

Figure 3. PPARβ/δ modulates epidermal differentiation via ANGPTL4.

(A) Immunoblot analysis of early (cytokeratin 10, CK 10), late (involucrin) and terminal (transglutaminase type I, Tgase 1) epidermal differentiation markers in K_CTRL, K_PPARβ/δ or K_ANGPTL4 keratinocytes. (B) Immunofluorescence staining of late epidermal differentiation (involucrin, INV) and proliferation (Ki67) markers of either K_CTRL-, K_PPARβ/δ- or K_ANGPTL4-dervied OTC sections, subjected to indicated treatments. PPARβ/δ agonist GW501516 (GW, 100 nM), PPARβ/δ antagonist GSK0660 (GSK, 0.5 µm), recombinant ANGPTL4 (rec. ANGPTL4, 6 µg/ml) and blocking ANGPTL4 monoclonal antibody (mAb11F6C4, 6 µg/ml). mAb11F6C4 targets the C-terminal fibrinogen-like domain of ANGPTL4 and has been shown to block ANGPTL4 interaction with integrin β1/β5. Immunoblot data are from three independent experiments performed in duplicate. β-tubulin serves as a loading and transfer control. White dotted lines indicated epidermis-dermis junctions. Sections were counterstained with DAPI (blue). Scale bars represent 40 µm.

Figure 4. ANGPTL4 modulates the expression of PKCs and activities of AP-1.

(A and C) Representative immunoblot analysis of indicated proteins from (A) epidermis of K_CTRL- and K_ANGPTL4-derived OTCs or (C) skin biopsies of ANGPTL4^+/+ and ANGPPTL4^−/− mice. Values below immunoblot bands represent the mean fold differences in protein expression levels when compared with either K_CTRL (for A) or ANGPTL4^+/+ (for C), which was assigned the value one, from 3 independent experiments. β-tubulin serves as a loading and transfer control. (B) Relative fold change in mRNA levels of c-JUN, PKCα and PKCδ in control (K_CTRL) human keratinocytes treated with either DMSO vehicle or recombinant ANGPTL4 (6 µg/ml), in the absence or presence of RNA synthesis (actinomycin D, Act-D) or protein synthesis (cycloheximide, CHX) inhibitors as determined by quantitative real-time PCR. Act-D and CHX treatment alone did not affect the transcript level. (D) Relative fold change in mRNA levels of ANGPTL4 and transglutaminase type I in human keratinocytes transiently transfected with expression vectors encoding for either MMK7-JNK or TAM67. Empty expression vector was used a scontrol. Ribosomal protein L27 was used as a normalizing reference gene. Values are mean ± SEM of three independent experiments. (E) Chromatin immunoprecipitations were done either with vehicle (PBS)- and recombinant ANGPTL4-treated K_ANGPTL4 or with K_CTRL and K_ANGPTL4 keratinocytes using pre-immune IgG (pre), antibody against phospho-cJUN and phospho-JUNB (Ab). The AP-1 binding site in the human transglutaminase type 1 and involucrin promoter region were immunoprecipitated and specifically amplified using phospho-cJUN and phospho-JUNB, respectively. No amplified signal was obtained in vehicle-treated K_ANGPTL4, K_ANGPTL4 or using pre-immune IgG. A control region upstream of the AP-1 binding site served as negative control. Aliquots of the chromatin were also analyzed before immunoprecipitation (input). M: 100-bp DNA marker.