Lipin-1 expression is critical for keratinocyte differentiation
2016; Elsevier BV; Volume: 57; Issue: 4 Linguagem: Inglês
10.1194/jlr.m062588
ISSN1539-7262
AutoresMinjung Chae, Ji-Yong Jung, Il‐Hong Bae, Hyoung-June Kim, Tae Ryong Lee, Dong Wook Shin,
Tópico(s)Cellular transport and secretion
ResumoLipin-1 is an Mg2+-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1. Lipin-1 is an Mg2+-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1. Lipin-1 was first described as the gene responsible for lipodystrophy in the fatty liver dystrophy mouse (1Péterfy M. Phan J. Xu P. Reue K. Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (468) Google Scholar). The function of lipin-1 as a mammalian Mg2+-dependent phosphatidate phosphatase was revealed by the identification of Pah1p, a yeast homolog, as an Mg2+-dependent phosphatidate phosphatase (2Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. 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Dotto G.P. p21WAF1/Cip1 suppresses keratinocyte differentiation independently of the cell cycle through transcriptional up-regulation of the IGF-I gene.J. Biol. Chem. 2006; 281: 30463-30470Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 33Okuyama R. LeFort K. Dotto G.P. A dynamic model of keratinocyte stem cell renewal and differentiation: role of the p21WAF1/Cip1 and Notch1 signaling pathways.J. Investig. Dermatol. Symp. Proc. 2004; 9: 248-252Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In this study, we investigated the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). We show that lipin-1 is present in basal and spinous layers of normal human epidermis, and that lipin-1 expression is gradually downregulated during NHEK differentiation. We also demonstrate that lipin-1 knockdown (KD) causes G1 arrest associated with p21 upregulation, which is known to be necessary for initial commitment of keratinocytes to differentiation. However, interestingly, lipin-1 KD suppressed keratinocyte differentiation. We demonstrate that the reduced keratinocyte differentiation was due to the sustained expression of p21 driven by lipin-1 KD, mediated by inhibition of PKCα activation. NHEKs from neonatal foreskin were purchased from Lonza (Basel, Switzerland) and cultured in KBM-GOLD medium with KGM-GOLD growth supplements containing insulin, human epidermal growth factor, bovine pituitary extract, hydrocortisone, epinephrine, transferrin, and gentamicin/amphotericin B. The cells were serially passaged at 70–80% confluence no more than three times. Normal human skin purchased from Biochain (Hayward, CA) was incubated with polyclonal rabbit anti-lipin-1 antibody (Sigma, St. Louis, MO) for 40 min at room temperature. Anti-rabbit HRP-conjugated antibody (ab6802; Abcam Inc., Cambridge, MA) was then applied as the secondary antibody for 20 min. The secondary antibody was detected with using chromogenic substrate 3,3′-diaminobenzidine. Immunoreactivity was evaluated under a light microscope (BX-53; Olympus, Tokyo, Japan) and photomicrographs were taken using a digital camera (DP72; Olympus). The human full-length lipin-1 cDNA (isoform 1α, accession number NM_145963) cloned into the pCMV6-AC-green fluorescent protein (GFP) vector was obtained from Origene (Rockville, MD). To generate lipin-1β isoform (accession number NM_001261428), lipin-1 fragment 1 (F1) was amplified by PCR from the genomic DNA template (forward, 5′-GAGGCAGACAGCACCATACA-3′ reverse, 5′-TCAATGGGCTGGACTCTTTC-3′). The DNA template was extended by four successive PCR reactions with first primers (forward, 5′-TGGATTCGAAATGTATGGTCTTCAAACATTAACGTGCAGACCATGAATTACGTGGGGCAG-3′ reverse, 5′-CTGCAAGATGAGGGGCAGTCCTTTTGCAATCTACCAGGCTACTGGGAGTGGGTGACCACTC-3′), second primers (forward, 5′-GACTCGGCTTGGTCATGGATTCCAATAATGAGAGACCCTGGGTGGATTCGAAATGTATGG-3′ reverse, 5′-GTGGAGGGCAAGAACTAGACAGACCTCCCTCGGCCGCAACTGCAAGATGAGGGGCAG-3′), third primers (forward, 5′-CGCAGCTCCAGCTGGGAGACCTCGCAGGGCAAGAGCTCCCCAGACTCGGCTTGGTCATGG-3′ reverse, 5′-GAACCGGAAGGACTTTCCGAAGGATGGAACAGGGAAGACTGTGGAGGGCAAGAACTAG-3′), and fourth primers (forward, 5′-AGATCTGCCGCCGCGATCGCCATGGGGGAACAGGACGGCATTCGCAGCTCCAGCTGGGAG-3′ reverse, 5′-GAACCGGAAGGACTTTCCGAAGGATGGAACAGGGAAGACTGTGGAGGGCAAGAACTAG-3′). The extended PCR product was amplified using primers with added restriction sites (forward, 5′-AGATCTGCCGCCGCGATCGCCATGGGGGAACAGGACGG-3′ reverse, 5′-GCGGCCGCGTACGCGTGTTAACAGGGAAGACTGTGG-3′), and the resultant lipin-1 fragment 2 (F2) was cloned into the pCMV6-AC-GFP vector at SgfI/MluI sites using the In-Fusion HD cloning kit (Clontech, Mountain View, CA). Lipin-1 fragment 3 (F3) was amplified by PCR from the genomic DNA template (forward, 5′-GAGGCAGACAGCACCATACA-3′ reverse, 5′-TCAATGGGCTGGACTCTTTC-3′) and the resultant fragment was cloned into the pCMV6-AC vector by inserting F2 at HpaI/MluI sites using the In-Fusion HD cloning kit. To prepare total cell lysate, NHEKs were washed with ice-cold PBS and lysed in RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40] in the presence of protease and phosphatase inhibitor cocktail (Sigma). The lysate was then centrifuged at 15,000 g for 20 min, and the supernatant was used for analysis. Protein concentration was determined using a BCA kit (Sigma) with BSA as the standard. Equal protein (40 μg/well) from cell lysates was loaded and separated by 8–12% gradient SDS-PAGE and transferred onto polyvinylidene difluoride membrane. Membranes were blocked in 3% BSA in TBST [20 mM Tris-HCl (pH 8.5), 150 mM NaCl, 0.5% Tween] at room temperature for 30 min. Blots were incubated at 4°C with anti-lipin-1 antibody (R&D Systems, Minneapolis, MN), and anti-cytokeratin 5, -cytokeratin 14, -cytokeratin 1, -cytokeratin 10 (Covance, Princeton, NJ), and anti-β actin, -GAPDH, -p21, -phospho myristoylated alanine-rich C kinase substrate (MARCKS) (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phospho PKCα (Ser657), -phospho PKCα (Thr638), -phospho PKCε (Ser729), -aquaporin 3, -involucrin (Abcam), and anti-phospho PKCδ (Thr505), -phospho PKCη (Ser674), -phospho PKCζ (Thr410), -phospho-serine PKC substrate, -MARCKS (Cell Signaling, Boston, MA) antibodies overnight in 3% BSA in TBST. Membranes were washed three times for 15 min in TBST followed by incubation with the appropriate HRP-conjugated goat anti-rabbit or rabbit anti-goat IgG secondary antibodies (Bio-Rad, Hercules, CA) for 1 h at room temperature. Membranes were washed and visualized with ECL immunofluorescence staining (Amersham Pharmacia Biotech, Piscataway, NJ). Image analysis of immunoblots was performed using ImageQuant TL software (GE Healthcare Life Sciences, Pittsburgh, PA). Predesigned ON-TARGET plus human siRNA against lipin-1 (#L01742701), p21 (#L00347100), and nontargeting pool siRNA (#D-001810-10) were purchased from Dharmacon (Lafayette, CO). NHEKs were plated 24 h before transfection and then transfected by lipofection using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) and OPTI-MEM (Invitrogen) with siRNA at a final concentration of 25 nM for 6 h. The medium was then changed to KGM-Gold containing all appropriate supplements. For lipin-1 overexpression, NHEKs were seeded 24 h before transfection and then transfected with 1 μg/ml of plasmid using the X-tremeGENE HP DNA transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. We used a 1.5:1 ratio of microliters of X-tremeGENE HP DNA transfection reagent to micrograms of pCMV6-AC-GFP vector as a transfection control. After cells were transfected with siRNAs, complete medium was replaced with serum-free medium for 16 h. The cells were centrifuged, washed in PBS, and fixed in 70% ethanol overnight at −20°C. After washing twice in PBS, the cells were stained in 0.5 ml of propidium iodide/RNase solution (BD Bioscience, San Jose, CA) for 15 min. Cell cycle distribution was analyzed using FACS Calibur (BD Bioscience). Ten thousand events were counted during data collection. The percentage of cells in G1, S, G2/M phase was determined using ModFit LT curve fitting software (Verity Software, Topsham, ME). Lipids were extracted by a modification of the Bligh and Dyer method (34Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42681) Google Scholar). Briefly, keratinocyte cells were collected by centrifugation at 1,500 g for 5 min and suspended in 0.5 ml of cold PBS buffer (pH 7.4) followed by sonication. Pellet was extracted with 1.5 ml of methanol, 2.25 ml of 1 M of sodium chloride, and 2.5 ml of chloroform, and the phase was separated by centrifugation at 1,500 g for 5 min. The lower phase was dried and redissolved in 500 μl of 1% Triton X-100 for PA analysis or 100 μl of chloroform for DAG analysis. PA was measured with the Total Phosphatidic Acid Fluorometric Assay kit (Cayman Chemical, Ann Arbor, MI) (10Zhang P. Verity M.A. Reue K. Lipin-1 regulates autophagy clearance and intersects with statin drug effects in skeletal muscle.Cell Metab. 2014; 20: 267-279Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). For the DAG quantification, extracted lipids in chloroform were spotted on silica 60 TLC plates (Sigma) (35Baldanzi G. Alchera E. Imarisio C. Gaggianesi M. Dal Ponte C. Nitti M. Domenicotti C. van Blitterswijk W.J. Albano E. Graziani A. et al.Negative regulation of diacylglycerol kinase theta mediates adenosine-dependent hepatocyte preconditioning.Cell Death Differ. 2010; 17: 1059-1068Crossref PubMed Scopus (25) Google Scholar). DAG was migrated with diethylether/heptane/acetic acid (75:25:1 v:v:v) mixture, the plates were dried and stained with 0.003% Coomassie brilliant blue in 30% methanol and 10 mM sodium chloride for 30 min. The plates were destained for 5 min in dye-free solution and the band density was calculated using ImageQuant TL software (GE Healthcare Life Sciences).The amount of DAG was calculated using a standard curve of 1,2-dioleyl-sn-diacylglycerol (Avanti Polar Lipids, Alabaster, AL) Statistical comparisons were performed using Student's t-test between two groups or one-way ANOVA test within multiple groups, followed by Turkey's post hoc test. All measurements were obtained from at least three independent experiments and values are expressed as the mean ± SD. To understand the physiological role of lipin-1 in keratinocytes, the lipin-1 protein level was measured during proliferation and differentiation. NHEKs were cultured in low calcium KGM-Gold medium up to 90% confluence (day 0). To study the regulation of lipin-1 protein expression by keratinocyte differentiation, NHEKs were cultured for an additional 7 days (day 7) under either low calcium (50 μM) or high calcium (1.2 mM) conditions. Western blot analysis showed that lipin-1 was strongly downregulated regardless of calcium concentration (Fig. 1A, B), corresponding to an increase in expression of involucrin, a marker for differentiation (29Missero C. Di Cunto F. Kiyokawa H. Koff A. Dotto G.P. The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.Genes Dev. 1996; 10: 3065-3075Crossref PubMed Scopus (284) Google Scholar, 31Di Cunto F. Topley G. Calautti E. Hsiao J. Ong L. Seth P.K. Dotto G.P. Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control.Science. 1998; 280: 1069-1072Crossref PubMed Scopus (253) Google Scholar). A previous report demonstrated that PKC activation mediates human keratinocyte differentiation at high cell densities, independent of changes in extracellular calcium (36Lee Y.S. Yuspa S.H. Dlugosz A.A. Differentiation of cultured human epidermal keratinocytes at high cell densities is mediated by endogenous activation of the protein kinase C signaling pathway.J. Invest. Dermatol. 1998; 111: 762-766Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), which may be associated with similar patterns of lipin-1 expression in low and high calcium conditions. The data indicate that lipin-1 expression is gradually decreased during keratinocyte differentiation. To assess the expression pattern and localization of lipin-1 in human epidermis, immunostaining was performed for lipin-1 in human foreskin sections. Lipin-1 was largely detected in both the basal layer and the spinous layer (Fig. 1C). Lipin-1 expression appeared to be purplish brown as a result of brown 3,3′-diaminobenzidine chromogen with blue hematoxylin. These data suggest that lipin-1 may play a role in keratinocyte proliferation or early differentiation. Because lipin-1 is predominantly expressed in proliferating keratinocytes, its effect on proliferation was examined by cell cycle analysis using propidium iodide staining. After transfection with control siRNA or lipin-1 siRNA, NHEKs were subjected to serum deprivation for 16 h and DNA content was analyzed by fluorescence-activated cell sorting. The DNA histogram revealed that the proportion of G1 phase was increased in a serum-free medium compared with control (43.03 ± 0.34% vs. 56.07 ± 0.24%) (see supplementary Fig. 1). Fluorescence-activated cell sorting analysis revealed that lipin-1 siRNA induced G1 cell-cycle arrest (56 ± 0.38%) 24 h after transfection compared with control siRNA (50.77 ± 0.57%; Fig. 2A). Analysis of proteins regulating the progression through the G1 phase of the cell cycle showed a strong increase in the levels of p21 and p53, but not p16 (Fig. 2C). Activation of p21 signaling is an important mechanism to prevent G1 cells from entering the S phase. The p21 is activated by p53-dependent or p53-independent mechanisms (37Abbas T. Dutta A. p21 in cancer: intricate networks and multiple activities.Nat. Rev. Cancer. 2009; 9: 400-414Crossref PubMed Scopus (1911) Google Scholar). Thus, we predict that lipin-1 KD results in an increased p53 level, which in turn induces its downstream target p21 expression and G1 arrest in NHEKs. Taken together, lipin-1 may be required for the progression of G1 phase in the cell cycle through regulating p53/p21 expression. To confirm DAG formation via dephosphorylation of PA by lipin-1 in keratinocytes, like in hepatocytes and adipocytes (1Péterfy M. Phan J. Xu P. Reue K. Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (468) Google Scholar, 8Csaki L.S. Dwyer J.R. Fong L.G. Tontonoz P. Young S.G. Reue K. Lipins, lipinopathies, and the modulation of cellular lipid storage and signaling.Prog. Lipid Res. 2013; 52: 305-316Crossref PubMed Scopus (93) Google Scholar, 9Pascual F. Carman G.M. Phosphatidate phosphatase, a key regulator of lipid homeostasis.Biochim. Biophys. Acta. 2013; 1831: 514-522Crossref PubMed Scopus (116) Google Scholar), PA and DAG contents were assayed in NHEKs transfected with lipin-1 or control siRNA. As expected, lipin-1 KD resulted in elevated PA and reduced DAG levels compared with control (Fig. 3A). The data indicated that lipin-1 is involved in DAG formation in NHEKs (supplementary Fig. 2). PKC activation by DAG controls the proliferation of keratinocytes (38Chew Y.C. Adhikary G. Wilson G.M. Reece E.A. Eckert R.L. Protein kinase C (PKC) delta suppresses keratinocyte proliferation by increasing p21(Cip1) level by a KLF4 transcription factor-dependent mechanism.J. Biol. Chem. 2011; 286: 28772-28782Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 39Jerome-Morais A. Rahn H.R. Tibudan S.S. Denning M.F. Role for protein kinase C-alpha in keratinocyte growth arrest.J. Invest. Dermatol. 2009; 129: 2365-2375Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To assess whether altered DAG contents led to impaired PKC activation, we assessed the phosphorylation of PKC substrates. Phosphorylation of PKC substrates and MARCKS, a major PKC substrate, was inhibited by lipin-1 KD (Fig. 3B–E). Phosphorylation of five PKC isoforms expressed in keratinocytes (11Osada K. Seishima M. Kitajima Y. Pemphigus IgG activates and translocates protein kinase C from the cytosol to the particulate/cytoskeleton fractions in human keratinocytes.J. Invest. Dermatol. 1997; 108: 482-487Abstract Full Text PDF PubMed Scopus (106) Google Scholar) was also analyzed by Western blot. Most PKC isoforms exhibited no significant change in phosphorylation levels in lipin-1 KD cells compared with control, but PKCα phosphorylation at Ser657 was inhibited by lipin-1 KD (Fig. 4A). To confirm the inhibition of PKCα activation, we demonstrated that lipin-1 KD decreased phosphorylation at another PKCα autophosphorylation site (Thr638) (Fig. 4C). We transfected vectors containing lipin-1 cDNA or pCMV-AC empty vector as a negative control to assess the effect of exogenous expression of lipin-1. Western blot analysis indicated that overexpression of lipin-1 enhances PKCα phosphorylation (Fig. 4E). These results indicate that lipin-1-catalyzed DAG formation is associated with the activation of PKC, specifically PKCα, in NHEKs.Fig. 4Lipin-1 regulates PKCα activity. A, B: Cell lysates after transfection of lipin-1 siRNA or control siRNA in NHEKs for 24 h were immunoblotted to assess phosphorylation levels of PKC is
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