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endoplasmic reticulum Ca2+ ATPase 2 heterozygote mice keratinocytes
Progress in Biophysics and Molecular Biology 103 (2010) 81e87
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Review
Markers of squamous cell carcinoma in sarco/endoplasmic reticulum Ca2þ ATPase 2 heterozygote mice keratinocytes Jeong Hee Hong a, Yu-Mi Yang a, Hyun Sil Kim b, Syng-Ill Lee a, Shmuel Muallem c, Dong Min Shin a, * a
Department of Oral Biology, Brain Korea 21 Project, Oral Science Research Center, Center for Natural Defense System, Yonsei University College of Dentistry, Seoul 120-752, Republic of Korea b Department of Oral Pathology, Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul 120-752, Republic of Korea c Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 17 October 2009
A mutation of Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca2þ-ATPase 2 (SERCA2) causes Darier's disease in human and null mutation in one copy of Atp2a2 leads to a high incidence of squamous cell tumor in a mouse model. In SERCA2 heterozygote (SERCA2þ/) mice keratinocytes, mechanisms involved in partial depletion of SERCA2 gene and its related tumor induction have not been studied. In this study, we investigated Ca2þ signaling and differential gene expression in primary cultured keratinocytes from SERCA2þ/ mice. SERCA2þ/ keratinocytes showed reduced initial increases in intracellular concentration of calcium in response to ATP, a G-protein coupled receptor agonist, and higher storeoperated Ca2þ entry with the treatment of thapsigargin, an inhibitor of SERCA, compared to wild type kerationcytes. Protein expressions of plasma membrane Ca2þ ATPases, NFATc1, phosphorylated ERK, JNK, and phospholipase g1 were increased in SERCA2þ/ keratinocytes. Using the gene fishing system, we first found in SERCA2þ/ keratinocytes that gene level of tumor-associated calcium signal transducer 1, crystalline aB, procollagen XVIII a1, and nuclear factor I-B were increased. Expression of involucrin, a marker of keratinocyte differentiation, was decreased in SERCA2þ/ keratinocytes. These results suggest that the alterations of Ca2þ signaling by SERCA2 haploinsufficiency alternate the gene expression of tumor induction and differentiation in keratinocytes. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Keywords: Ca2þ signaling SERCA2þ/ Squamous cell carcinoma Keratinocytes
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.2. Primary keratinocyte culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.3. Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.4. Intracellular concentration of Ca2þ ([Ca2þ]i) measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.5. Immunoblotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 2.6. First-strand cDNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 2.7. ACP-based GeneFishingÔ PCR for second strand synthesis and direct sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 2.8. Quantitative real-time RT-PCR (Q-rtPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 2.9. Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 3.1. Squamous cell carcinomas were induced in perineal skin of SERCA2þ/ mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 3.2. Altered Ca2þ signaling in SERCA2þ/ keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Abbreviations: SERCA2, sarco/endoplasmic reticulum Ca2þ-ATPase 2; SCC, squamous cell carcinoma; [Ca2þ]i, intracellular concentration of calcium; TGF b, transforming growth factor b; PSS, physiologic salt solution; DEGs, differentially expressed genes; SOC, store-operated Ca2þ influx; Tg, thapsigargin; PLC g, phospholipase C g; NFI-B, nuclear factor I-B; PDGFb, platelet-derived growth factor b; IL, interleukin. * Corresponding author. Tel.: þ82 2 2228 3051; fax: þ82 2 364 1085. E-mail address: [email protected] (D.M. Shin). 0079-6107/$ e see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.pbiomolbio.2009.10.005
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3.3. Patterns of cancer-related signaling proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 3.4. Patterns of differentially expressed genes (DEGs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
1. Introduction
2. Material and methods
Skin cancer is the most common type of human cancer that can metastasize and lead to death. Squamous Cell Carcinomas (SCCs), which arise in multilayered epithelia such as the epidermis, cervix, lip, tongue, and floor of the mouth (Janes and Watt, 2006), are the most common type of oral cancers and more than 90% of the reported SCCs are malignant (Chen et al., 2004). It has been suggested that SCCs of the skin increase through a variety of process that involve activation of proto-oncogenes and/or inactivation of tumor suppressor genes by ultraviolet irradiation (UV), UV-induced oxidative stress (Melnikova and Ananthaswamy, 2005), inflammation (Li et al., 2006), DNA damage (Berhane et al., 2002). p53 heterozygosity was found to be correlated with tumor induction in a mouse model (Rebel et al., 2001). Loss of transforming growth factor b (TGFb) type II receptor and the overexpression of K-ras or H-ras have been shown to induce SCC of the head and neck (Lu et al., 2006). In addition, disruption of a variety of intracellular cell signalings related to extracellular signaling is involved in the generation of SCCs. Defective Ca2þ homeostasis causes skin diseases such as Darier's disease (DD) (Ahn et al., 2003; Foggia and Hovnanian, 2004; Sakuntabhai et al., 1999; Zhao et al., 2001), which is an autosomal dominant skin disorder characterized by multiple keratotic papules caused by a null mutation in one copy of the Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca2þ ATPase (SERCA2) (Sakuntabhai et al., 1999). The SERCA2 pumps are responsible for loading the endoplasmic reticulum (ER) Ca2þ and maintaining the Ca2þ gradients between the cytosol and the lumen of the ER (GunteskiHamblin et al., 1988). It has been suggested that the ER Ca2þ content contributes to the maintenance and the differentiated epidermis, including skin barrier function (Bikle et al., 2001; Elias et al., 2002). Loss of ER Ca2þ causes increased cell proliferation and decreased expression of differentiation markers, such as involucrin, filaggrin, and loricrin (Bikle et al., 2001). The SERCA2b is exclusively expressed in keratinocytes (Pani et al., 2006) and inhibition of SERCA2 with thapsigargin (Tg), an inhibitor of SERCA, disrupts a variety of biological processes, particularly during terminal differentiation of keratinocytes (Lowry et al., 1996). Moreover, the loss of one copy of the Atp2a2 allele induced the formation of SCCs in SERCA2 heterozygote (SERCA2þ/) mice (Liu et al., 2001). SERCA2þ/ mice show enhanced tumor susceptibility that is followed by tumor initiation and progression via elevated expression of wild type H-ras, K-ras, and p53 in SERCA2þ/ (Prasad et al., 2005), which suggests that aberrant Ca2þ signaling due to SERCA2 haploinsufficiency is associated with susceptibility to carcinogenesis. SERCA2þ/ mice serve as an animal model for skin tumors (Liu et al., 2001; Prasad et al., 2005). However, in SERCA2þ/ mice keratinocytes, the event of Ca2þ signaling and its related tumor induction and keratinocyte differentiation have not been studied and the role of intracellular Ca2þ in the overall cancerrelated signaling pathway is also uncontested in the primary cultured keratinocyte. In the present work, we investigated the expression of tumorigenic factors in primary cultured keratinocytes as well as the effect of altered SERCA2 expression on Ca2þ signaling.
2.1. Materials Trypsin, ATP, DNase, Hanks' balanced salt solution (HBSS), trypsin, type I collagen, and collagen were purchased from Sigma; Defined keratinocyte media, gentamycin, penicillin, streptomysin, thapsigargin, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA); fura-2 acetoxymethyl ester (fura-2/AM) was purchased from Teflabs (Austin, TX). All other chemicals were used reagent grade. 2.2. Primary keratinocyte culture Wild type (WT) and SERCA2þ/ mice in a Black Swiss background were kindly gifts from Dr. Gary E Shull (University of Cincinnati College of Medicine, USA) (Periasamy et al., 1999), and housed with free access to food and water in a temperaturecontrolled room (23 1 C) under artificial illumination (lights on 06:00 he18:00 h) and 55% relative humidity. All animal protocols were performed according to institutional guidelines of Yonsei University College of Dentistry. Genotypes were determined by PCR analysis of tail DNA as described previously (Periasamy et al., 1999). Dorsal and ventral skin of one mouse was used for keratinocytes culture. The skins were carefully shaved off all body hair, stretched, tissue debris was removed carefully with a scrapper, and the skins were floated on 0.5% trypsin in HBSS for 25 min at 37 C. The floated epidermis was carefully separated from the dermis, neutralized by HBSS including 0.05% DNase and 20% fetal bovine serum, and minced with scissors. The suspension was filtrated through autoclaved nylon nets to take off the remaining body hair and tissue fragments. After sedimentation, the epidermal cells were washed with HBSS, centrifuged for 5 min at 1000 rpm, and re-suspended gently in defined keratinocyte media including 5 mg/ml gentamycin. The cells were cultured on type I collagen-coated dishes in an incubator at 5% CO2 and 37 C and used at 80% confluency. The media was periodically changed every 2e3 days. 2.3. Histology Mouse skins from the lip and genitalia were fixed in 4% paraformaldehyde in phosphate-buffed saline overnight at 4 C, and embedded in paraffin wax. Images of 6 mm thick paraffin sections stained with hematoxylin and eosin (H & E) were obtained using a Leica microscope (Germany). 2.4. Intracellular concentration of Ca2þ ([Ca2þ]i) measurement [Ca2þ]i was determined in primary mouse keratinocytes by fura2/AM in an extracellular physiologic salt solution (PSS), the composition of which was as follows (mM): 140 NaCl, 5 KCl, 5 mM; 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose, titrated to pH 7.4 with NaOH. The osmolarity of the PSS was 310 mOsm. Ca2þ-free medium contained 1 mM EDTA and 1 mM ethyleneglycol-bis-(b-aminoethylether)-N,N,N0 ,N0 -tetra acetic acid (EGTA) in PSS. The primary keratinocytes were cultured on collagen-coated cover glasses for
J.H. Hong et al. / Progress in Biophysics and Molecular Biology 103 (2010) 81e87
measuring [Ca2þ]i. The cells were loaded with 3 mM fura-2/AM for 1 h and after washing with standard solution, [Ca2þ]i was measured by alternately illuminating the cells at wavelengths 340 and 380 nm, and the emitted light was passed through a 510 nm cutoff filter and was collected with a CCD camera and analyzed with a MetaFluor system (Universal Imaging Co., Downingtown, PA). The 340/380 fura-2 ratio was taken as a measure of [Ca2þ]i and fluorescence images were obtained at 3 s intervals. 2.5. Immunoblotting Cells were lysed by adding RIPA (radio-immuno precipitation assay) buffer containing in mM; 10 TriseHCl (pH 7.8), 150 NaCl, 1 EDTA, 1% NP-40, 10 Na3VO4, 10 NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 10 mg/ml PMSF. Lysates were centrifuged at 11,000 rpm for 10 min, and supernatants were collected for immunoblotting. Protein concentration was determined using the BCA assay kit (Pierce, IL). Equal protein amounts (40 mg) were separated by 10% SDS-PAGE gel (Bio-Rad, CA) and electro-transferred onto nitrocellulose membranes. The membranes were then incubated in 5% skim milk powder in TBST (mM); 20 TriseHCl, (pH 7.6), 137 NaCl, and 0.1% Tween 20 for 1 h, and incubated sequentially with primary antibody and followed by horseradish peroxidase-conjugated secondary antibody (SantaCruz, CA). Blotted proteins were visualized by an enhanced chemiluminescence reagent (IntRON, South Korea). 2.6. First-strand cDNA synthesis Total RNA was extracted from cultured keratinocytes and was used for the synthesis of first-strand cDNAs by reverse transcriptase. Reverse transcription was performed for 1.5 h at 42 C in a final reaction volume of 20 ml containing 3 mg of the purified total RNA, 4 ml of 5 reaction buffer (Promega, Madison, WI), 5 ml of dNTPs (each 2 mM), 2 ml of 10 mM dT-ACP1 (50 -CTGTGAATGCTGC GACTACGATIIIIIT-30 ), 0.5 ml of RNasinÒ RNase Inhibitor (Promega), and 1 ml of Moloney murine leukemia virus reverse transcriptase (Promega). First-strand cDNAs were diluted by the addition of 80 ml of purified water for RT-PCR.
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normalized b2M in WT and SERCA2þ/ samples. The data were analyzed with the Thermal Cycler DiceÔ Real Time System analysis software (Takara). The primer sequences of genes are as follows (forward/reverse): tumor-associated calcium signal transducer 1 (TACSTD1, 50 -TTGTGGTGGTGTCATTAGCAGTCA-30 /50 -CACTCAGCAC GGCTAGGCATTA-30 ), crystalline aB (50 -TGATTGAGGTCCACGGCAA G-30 /50 -ACAGTGAGGACTCCATCAGATGACA-30 ), procollagen type XVIII a1 (50 -GTGCCCATCGTCAACCTGAA-30 /50 -AGTTGACCCTGGGA GCCAGA-30 ), nuclear factor I-B (NFI-B, 50 -AATACCTGGAGTCGCG CACA-30 /50 -GGATAGCTTGCGTCGGAAATG-30 ), involucrin (50 -GCAA GACATGCTAGTACCACAGGAG-30 /50 -CTGCTGACCCAGATGCAGTTC-30 ), and b-2 microglobulin (b2M, 50 -GGGAAGCCGAACATACTGAA-30 /50 TCACATGTCTCGATCCCAGT-30 ). The data were expressed as mean SD. The mean expression levels were compared between WT and SERCA2þ/ mice group using ANOVA (SPSS). 2.9. Statistics All data were given as mean SE. Statistical significances of between groups were determined using the Student's t-test. 3. Results 3.1. Squamous cell carcinomas were induced in perineal skin of SERCA2þ/ mice Cancer, which was first observed in 22-week-old SERCA2þ/ mice, reached 100% incidence by the time the mice were 52-week of age. Skin samples were removed from whole body of 52-weekold SERCA2þ/ mice and analyzed by H & E staining. Microscopic view showed the skin lesion was composed of tumor cell nests (T) invading the connective tissues in SERCA2þ/ mice (Fig. 1A). Highpower view of the lesion exhibit common features of cellular atypia with pleomorphism, hyperchromatism, and aberrant accumulations of keratin (arrow) in SERCA2þ/ mice (Fig. 1B). We hypothesized that modulation of Ca2þ signal involved in partial depletion of SERCA2 gene was occurred and which types of genes were involved in SERCA2þ/ keratinocytes. To perform primary keratinocytes culture in subsequent experiments, we used these skin lesions.
2.7. ACP-based GeneFishingÔ PCR for second strand synthesis and direct sequencing
3.2. Altered Ca2þ signaling in SERCA2þ/ keratinocytes
Differentially expressed genes (DEGs) were screened by ACPbased PCR method (Kim et al., 2004) using the GeneFishingÔ DEG kits (Seegene, South Korea). The PCR for second strand synthesis was performed according to the manufacturer's protocol. The amplified PCR products were separated in 2% agarose gel stained with ethidium bromide. Expression levels of DEGs were calculated by the MetaMorph system (Universal imaging Co., Downingtown, PA). The bands of the DEGs were re-amplified and extracted from the gel using the GENCLEANÒII Kit (Q-BIO gene, CA), and directly sequenced with ABI PRISMÒ3100-AvantGenetic Analyzer (Applied Biosystems, CA).
Fig. 2A and B shows that the level of SERCA2b protein was 31.2 1.1% lower in SERCA2þ/ than in WT mice (n ¼ 4). All analyses were conducted on mice that were more than 6 weeks of age to ensure that the epidermal layer was sufficiently mature. Densitometric analysis of the results revealed that the level of PMCA protein was 4.54 0.89 fold higher in SERCA2þ/ than in WT keratinocytes (Fig. 2A and B, n ¼ 4). The Ca2þ signal of keratinocytes obtained from the two mice strains was then evaluated by measuring [Ca2þ]i. The agonistevoked Ca2þ signal was triggered by stimulation of the native P2Y2 receptors with high concentration of ATP (Fig. 2C, n ¼ 10). The ATPinduced maximum increase of [Ca2þ]i of cells maintained in media containing 1 mM extracellular Ca2þ was reduced by 79.4 2.1% in SERCA2þ/ keratinocytes, as reflected in the reduction in the 340/ 380 ratio (Fig. 2E, left panel). Store-operated Ca2þ influx (SOC) was assayed by passive depletion of the stores by inhibition of the SERCA pumps with thapsigargin (Tg) and incubating the cells in nominally Ca2þ-free medium (Fig. 2D, n ¼ 5) for 15 min. Tg-triggered Ca2þ release was lower by 55.1 0.7% in SERCA2þ/ than in WT keratinocytes. SOC activity was 1.58 0.32 fold higher in SERCA2þ/ keratinocytes than in WT keratinocytes (Fig. 2E, right panel).
2.8. Quantitative real-time RT-PCR (Q-rtPCR) Total RNA was isolated the primary cultured keratinocyte cells using Trizol (Invitrogen) reagent. cDNA synthesis was performed using M-MLV reverse transcriptase for reverse transcription (Invitrogen). Quantitative real-time PCR was performed with SYBR premix EX Taq polymerase (Takara) using Thermal Cycler DiceÔ Real Time System (TP800, Takara). The amplification condition was 45 cycles of denaturation at 95 C for 5 s, annealing at 55 C for 20 s, and extension step at 72 C for 20 s. The results of Q-rtPCR were
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Fig. 1. Squamous cell carcinomas were induced in perineal skin of SERCA2þ/ mice. A. The skin lesion of perineum was composed of tumor cell nests (T) invading the connective tissues (H & E, original magnification 40), Indicated scale bar: 1000 mm B. High-power view of the lesion with pleomorphism, hyperchromatism, and aberrant accumulations of keratin (arrow) (H & E, original magnification 200), Indicated scale bar: 100 mm.
Fig. 2. Expressions of Ca2þ signaling proteins and Ca2þ signaling in primary keratinocytes from wild type and SERCA2þ/ mice. A and B, show protein (40 mg) levels of SERCA2b, PMCA, and b-actin in primary keratinocytes. b-actin was used for immunoblotting control. C, Primary keratinocytes obtained from wild type (WT, solid trace) and SERCA2þ/ (dotted trace) mice were stimulated with 100 mM ATP in PSS. D, Cells were perfused with Ca2þ-free medium (open bar) and then treated with 1 mM thapsigargin (Tg) in Ca2þ-free medium for 15 min. After completion of store depletion Ca2þ influx was assayed by the addition of 1 mM Ca2þ to the perfusion media (marked by dark bar). E, Maximum increase in [Ca2þ]i as revealed by the increase in Fura-2 (Rmax-R0) ratio in response to ATP stimulation (left panel). The rate of Ca2þ entry obtained from the period of Ca2þ addition to cells with depleted stores, as in panel D (right panel). Results are depicted as mean S.E.
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Fig. 3. Effect of disrupted Ca2þ signaling on expression of cancer-related signaling proteins. A, show protein level of phospho-ERK (P-ERK), total-ERK (T-ERK), phospho-JNK (P-JNK), total-JNK (T-JNK), phospho-PLCg1 (P-PLCg1), total-PLCg1 (T-PLCg1), NFATc1, and b-actin in primary cultured keratinocytes. B shows the summary of multiple experiments with n 4.
Fig. 4. Patterns of differentially expressed genes (DEGs) in response to SERCA2 haploinsufficiency. A, Relative expression level of ACP-based differentially expressed genes. B, Relative fluorescence unit of 5 genes in 42-week-old mice (n ¼ 4, pairs) using Quantitative real-time PCR. b2M, housekeeping gene, was used mRNA loading control. C, mRNA levels of cytokine-related DEGs. P; positive control, 1; WT, 2; SERCA2þ/, Housekeeping genes (SDHA, HPRT1, and b2M) were used as mRNA controls.
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3.3. Patterns of cancer-related signaling proteins
4. Discussion
Alteration of Ca2þ signaling in Fig. 2 can affect most of cancerrelated signaling pathways. Therefore, we hypothesized in SERCA2þ/ keratinocytes that the activities of caner-related molecules might be changed. The activities of ERK, JNK, phospholipase Cg1 (PLCg1), and NFATc1 in SERCA2þ/ keratinocytes were measured. The steady-state level of the expression of phospho-JNK protein and phospho-ERK protein was increased by 1.37 0.04 and 2.05 0.47 fold in SERCA2þ/ keratinocytes, respectively (n ¼ 4). The level of phospho-PLCg1 protein was 3.06 0.58 fold higher in SERCA2þ/ than in WT keratinocytes (Fig. 3A and B). In addition, the expression of NFATc1 was 3.24 0.09 fold higher in SERCA2þ/ than in WT keratinocytes.
In the present work we found a critical role of Ca2þ signaling in tumor induction and keratinocyte differentiation. Reduction in SERCA2 activity increased both SOCs-mediated Ca2þ influx and PMCA expression in keratinocytes (Fig. 2A). Similar results of SOCsmediated Ca2þ influx had been reported (Pani et al., 2006) that disruption of ER Ca2þ content is associated with an increase in TRPC1 expression in epidermal skin cells from Darier's disease patients and up-regulation of TRPC1 arguments cell proliferation and restrict apoptosis (Bollimuntha et al., 2005, 2006). For the protective role in epithelial layer, keratinocyte differentiation not proliferation plays a crucial role as a normal skin function. Both induction of differentiation and inhibition of proliferation are controlled by Ca2þ increase. Therefore, higher Ca2þ entry across the plasma membrane is important to determine cell fate such as differentiation, proliferation, tumor induction, etc. (Fig. 2D). Prevarskaya et al. (2007) showed the differential role of TRP channels in Ca2þ signal homeostasis of prostate cancer epithelial cells and their potential role in prostate carcinogenesis. Although the key determinants of Ca2þ homeostasis in skin cancer have been outlined, obviously it is important in cancer-associated growth and cell death regulation. Another change in Ca2þ signaling protein was increased phosphorylation of PLCg1, which is likely to increase cellular excitability to maintain the Ca2þ signaling pathway in a more active state under resting condition (Fig. 3). In addition, the expression of the Ca2þregulated transcription factor NFATc1 was markedly increased in SERCA2þ/ keratinocytes (Fig. 3). MAPKs such as JNK, ERK, and p38 are important regulatory proteins that transduce various extracellular signals into intracellular events (Seger and Krebs, 1995) and these MAPKs are modulated by disruption of ER Ca2þ homeostasis (Liang et al., 2006), and ERK activation is an important downstream effecter mechanism for cellular protection from ER stress (Hung et al., 2003). Therefore, the present results of ERK and JNK activation suggest that dysfunction of Ca2þ homeostasis caused by a partial loss of SERCA2 in SERCA2þ/ karatinocytes is related to events of remodeling of the Ca2þ signal-related protein, such as activation of PLCg1, increased SOCs and increased PMCA that activates cell excitability. The most notable work of present study is up-regulated DEGs and down-regulated DEGs in keratinocytes correspond to a variety of carcinogenesis signals (Table 1A and B). For example, TACSTD is involved in the early stages of human lung adenocarcinogenesis (Shimada et al., 2005). The results of our study revealed that in keratinocytes TACSTD1 might be involved in tumor induction. Collagen type XVIII expression is a useful prognostic marker in patients with non-small cell lung carcinoma (Chang et al., 2004) and NFI-B gene is related to tumor generation via fusion (Gronostajski, 2000). Finally, Crystallin aB has chaperone activities in its functional role of holding or folding multiple proteins that have been denatured simultaneously under stress conditions (Chen et al., 2004; Ohto-Fujita et al., 2007). Although it is unclear if the mechanism of TACSTD, NFI-B, procollagen type XVIII, and crystalline aB are related to SERCA2 haploinsufficiency, these up-regulated genes may affect tumor induction in keratinocytes. Down-regulation of involucrin in SERCA2þ/ keratinocyte is related to the differentiation of keratinocytes (Table 1B). Obviously, abnormal Ca2þ signal by SERCA2 depletion inhibits the mechanism of keratinocyte differentiation. This report suggests that disorder of the SERCA2 pump potentially disrupts to build normal skin structure. The TGFbs are multifunctional cytokines families that play a pivotal role in the maintenance of tissue homeostasis. TGFb1 overexpression in the basal layer of the epidermis and hair follicles causes a severe inflammatory skin disorder and epidermal
3.4. Patterns of differentially expressed genes (DEGs) Increased activities of cancer-related signaling proteins in Fig. 3 are likely to affect the expression of cell cycle and other tumorrelated genes (Schubbert et al., 2007). To search for genes affected by the partial deletion of SERCA2 and its downstream-related pathways, the mRNA pool expressed in 52-week-old WT and SERCA2þ/ keratinocytes (n ¼ 4) was compared using an ACP-based gene fishing PCR method. With this approach we found 5 genes that were differentially expressed between the both types of keratinocytes (Fig. 4A). The DEGs that showed the greatest differences were selected for direct sequencing, and the sequences were then evaluated using BLAST searches of the GeneBank database (Table 1). Genes that were increased in SERCA2þ/ keratinocytes included tumor-associated calcium signal transducer 1 (TACSTD1), crystalline aB, nuclear factor I-B (NFI-B), and procollagen XVIII a1, whereas involucrin was decreased in SERCA2þ/ keratinocytes. To confirm these gene profiles, Q-rtPCR was performed using above 5 target primers in 42-week-old mice (Fig. 4B, n ¼ 4, pairs). Cancer cells are capable of attracting different cell types into the tumor environment through angiogenic factors and cytokines (Schubbert et al., 2007; Shchors and Evan, 2007). Therefore, to determine which cytokines were related to the cutaneous tumor induction by SERCA2 haploinsufficiency, ACP-based cytokine PCR was performed. Fig. 4C shows the expression patterns of cytokines in 52-week-old WT and SERCA2þ/ keratinocytes (n ¼ 4). None of the known housekeeping genes analyzed (SDHA; HPRT1, and b2M) was changed in SERCA2þ/ keratinocytes. The expression of TGFb1 and 3 as well as platelet-derived growth factor b (PDGFb), interleukin (IL)-11, and IL-16 were increased in SERCA2þ/ keratinocytes. These results show that partial depletion of SERCA2 can be a potent inducer of cytokines causing cutaneous tumor.
Table 1 Identification of differentially expressed transcripts in response to SERCA2 haploinsufficiency. Identity A. Up-regulation Crystallin aB Nuclear factor I-B Procollagen type XVIII a1 Tumor-associated calcium signal transducer 1 B. Down-regulation Involucrin
GenBank Acc. No.
Base pairs
Sequence homology
BC BC BC BC
848 2637 5029 1492
100% 100% 100% 100%
1902
99% (369/370)
010768 096542 064817 005618
NM 008412
(477/477) (596/596) (585/585) (575/575)
* The percentage expressions are based on BLAST searches of the GenBank databases. The numbers in brackets show the number of bases (query/subject) that were compared.
J.H. Hong et al. / Progress in Biophysics and Molecular Biology 103 (2010) 81e87
carcinogenesis (Li et al., 2006). IL-11 is a pleiotropic cytokine that exhibits anti-inflammatory properties (Theodore et al., 1996), and the inhibition of allogenic lymphocyte reactivity might be due to the direct action of secreted IL-16 (Fujita et al., 2000). The results of the present study shows that SERCA2 haploinsufficiency and perturbation of Ca2þ signaling are powerful signals that induce abnormal expression of the TGFb1 and IL-11 genes (Fig. 4C). However, we presently do not understand how perturbed Ca2þ signal modulate these genes in keratinocytes. In conclusion, our findings show that haploinsufficiency of SERCA2 results in altered intracellular Ca2þ levels and adaptation of the Ca2þ signaling machinery to activate cell excitability. It subsequently evokes the switch-on mechanism of oncogenes expression and the switch-off mechanism of keratinocyte differentiation. Although the precise mechanisms by which cutaneous tumor induction occurs remain to be elucidated, the results of this study demonstrate that SERCA2 haploinsufficiency is linked to the promotion of these genetic abnormalities and may be crucial to tumor induction, at least in a subset of SCCs. Acknowledgements We thank Dr. Gary E Shull for offering the wild-type and SERCA2þ/ mice in a Black Swiss background. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0001658, 20100000315). References Ahn, W., Lee, M.G., Kim, K.H., Muallem, S., 2003. Multiple effects of SERCA2b mutations associated with Darier's disease. J. Biol. Chem. 278, 20795e20801. Berhane, T., Halliday, G.M., Cooke, B., Barnetson, R.S., 2002. Inflammation is associated with progression of actinic keratoses to squamous cell carcinomas in humans. Br. J. Dermatol. 146, 810e815. Bikle, D.D., Ng, D., Tu, C.L., Oda, Y., Xie, Z., 2001. Calcium- and vitamin D-regulated keratinocyte differentiation. Mol. Cell Endocrinol. 177, 161e171. Bollimuntha, S., Ebadi, M., Singh, B.B., 2006. TRPC1 protects human SH-SY5Y cells against salsolinol-induced cytotoxicity by inhibiting apoptosis. Brain Res. 1099, 141e149. Bollimuntha, S., Singh, B.B., Shavali, S., Sharma, S.K., Ebadi, M., 2005. TRPC1mediated inhibition of 1-methyl-4-phenylpyridinium ion neurotoxicity in human SH-SY5Y neuroblastoma cells. J. Biol. Chem. 280, 2132e2140. Chang, H., Iizasa, T., Shibuya, K., Iyoda, A., Suzuki, M., Moriya, Y., Liu, T.L., Hiwasa, T., Hiroshima, K., Fujisawa, T., 2004. Increased expression of collagen XVIII and its prognostic value in nonsmall cell lung carcinoma. Cancer 100, 1665e1672. Chen, J., He, Q.Y., Yuen, A.P., Chiu, J.F., 2004. Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis. Proteomics 4, 2465e2475. Elias, P.M., Ahn, S.K., Denda, M., Brown, B.E., Crumrine, D., Kimutai, L.K., Komuves, L., Lee, S.H., Feingold, K.R., 2002. Modulations in epidermal calcium regulate the expression of differentiation-specific markers. J. Invest. Dermatol.119, 1128e1136. Foggia, L., Hovnanian, A., 2004. Calcium pump disorders of the skin. Am. J. Med. Genet. C Semin. Med. Genet. 131C, 20e31. Fujita, T., Matsumoto, Y., Hirai, I., Ezoe, K., Saito, T., Yagihashi, A., Torigoe, T., Homma, K., Takahashi, S., Cruikshank, W.W., Jimbow, K., Sato, N., 2000. Immunosuppressive effect on T cell activation by interleukin- 16-cDNA-transfected human squamous cell line. Cell Immunol. 202, 54e60. Gronostajski, R.M., 2000. Roles of the NFI/CTF gene family in transcription and development. Gene 249, 31e45. Gunteski-Hamblin, A.M., Greeb, J., Shull, G.E., 1988. A novel Ca2þ pump expressed in brain, kidney, and stomach is encoded by an alternative transcript of the slowtwitch muscle sarcoplasmic reticulum Ca-ATPase gene. Identification of cDNAs
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Review
Markers of squamous cell carcinoma in sarco/endoplasmic reticulum Ca2þ ATPase 2 heterozygote mice keratinocytes Jeong Hee Hong a, Yu-Mi Yang a, Hyun Sil Kim b, Syng-Ill Lee a, Shmuel Muallem c, Dong Min Shin a, * a
Department of Oral Biology, Brain Korea 21 Project, Oral Science Research Center, Center for Natural Defense System, Yonsei University College of Dentistry, Seoul 120-752, Republic of Korea b Department of Oral Pathology, Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul 120-752, Republic of Korea c Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 17 October 2009
A mutation of Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca2þ-ATPase 2 (SERCA2) causes Darier's disease in human and null mutation in one copy of Atp2a2 leads to a high incidence of squamous cell tumor in a mouse model. In SERCA2 heterozygote (SERCA2þ/) mice keratinocytes, mechanisms involved in partial depletion of SERCA2 gene and its related tumor induction have not been studied. In this study, we investigated Ca2þ signaling and differential gene expression in primary cultured keratinocytes from SERCA2þ/ mice. SERCA2þ/ keratinocytes showed reduced initial increases in intracellular concentration of calcium in response to ATP, a G-protein coupled receptor agonist, and higher storeoperated Ca2þ entry with the treatment of thapsigargin, an inhibitor of SERCA, compared to wild type kerationcytes. Protein expressions of plasma membrane Ca2þ ATPases, NFATc1, phosphorylated ERK, JNK, and phospholipase g1 were increased in SERCA2þ/ keratinocytes. Using the gene fishing system, we first found in SERCA2þ/ keratinocytes that gene level of tumor-associated calcium signal transducer 1, crystalline aB, procollagen XVIII a1, and nuclear factor I-B were increased. Expression of involucrin, a marker of keratinocyte differentiation, was decreased in SERCA2þ/ keratinocytes. These results suggest that the alterations of Ca2þ signaling by SERCA2 haploinsufficiency alternate the gene expression of tumor induction and differentiation in keratinocytes. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Keywords: Ca2þ signaling SERCA2þ/ Squamous cell carcinoma Keratinocytes
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.2. Primary keratinocyte culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.3. Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.4. Intracellular concentration of Ca2þ ([Ca2þ]i) measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 2.5. Immunoblotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 2.6. First-strand cDNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 2.7. ACP-based GeneFishingÔ PCR for second strand synthesis and direct sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 2.8. Quantitative real-time RT-PCR (Q-rtPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 2.9. Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 3.1. Squamous cell carcinomas were induced in perineal skin of SERCA2þ/ mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 3.2. Altered Ca2þ signaling in SERCA2þ/ keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Abbreviations: SERCA2, sarco/endoplasmic reticulum Ca2þ-ATPase 2; SCC, squamous cell carcinoma; [Ca2þ]i, intracellular concentration of calcium; TGF b, transforming growth factor b; PSS, physiologic salt solution; DEGs, differentially expressed genes; SOC, store-operated Ca2þ influx; Tg, thapsigargin; PLC g, phospholipase C g; NFI-B, nuclear factor I-B; PDGFb, platelet-derived growth factor b; IL, interleukin. * Corresponding author. Tel.: þ82 2 2228 3051; fax: þ82 2 364 1085. E-mail address: [email protected] (D.M. Shin). 0079-6107/$ e see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.pbiomolbio.2009.10.005
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4.
J.H. Hong et al. / Progress in Biophysics and Molecular Biology 103 (2010) 81e87
3.3. Patterns of cancer-related signaling proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 3.4. Patterns of differentially expressed genes (DEGs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
1. Introduction
2. Material and methods
Skin cancer is the most common type of human cancer that can metastasize and lead to death. Squamous Cell Carcinomas (SCCs), which arise in multilayered epithelia such as the epidermis, cervix, lip, tongue, and floor of the mouth (Janes and Watt, 2006), are the most common type of oral cancers and more than 90% of the reported SCCs are malignant (Chen et al., 2004). It has been suggested that SCCs of the skin increase through a variety of process that involve activation of proto-oncogenes and/or inactivation of tumor suppressor genes by ultraviolet irradiation (UV), UV-induced oxidative stress (Melnikova and Ananthaswamy, 2005), inflammation (Li et al., 2006), DNA damage (Berhane et al., 2002). p53 heterozygosity was found to be correlated with tumor induction in a mouse model (Rebel et al., 2001). Loss of transforming growth factor b (TGFb) type II receptor and the overexpression of K-ras or H-ras have been shown to induce SCC of the head and neck (Lu et al., 2006). In addition, disruption of a variety of intracellular cell signalings related to extracellular signaling is involved in the generation of SCCs. Defective Ca2þ homeostasis causes skin diseases such as Darier's disease (DD) (Ahn et al., 2003; Foggia and Hovnanian, 2004; Sakuntabhai et al., 1999; Zhao et al., 2001), which is an autosomal dominant skin disorder characterized by multiple keratotic papules caused by a null mutation in one copy of the Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca2þ ATPase (SERCA2) (Sakuntabhai et al., 1999). The SERCA2 pumps are responsible for loading the endoplasmic reticulum (ER) Ca2þ and maintaining the Ca2þ gradients between the cytosol and the lumen of the ER (GunteskiHamblin et al., 1988). It has been suggested that the ER Ca2þ content contributes to the maintenance and the differentiated epidermis, including skin barrier function (Bikle et al., 2001; Elias et al., 2002). Loss of ER Ca2þ causes increased cell proliferation and decreased expression of differentiation markers, such as involucrin, filaggrin, and loricrin (Bikle et al., 2001). The SERCA2b is exclusively expressed in keratinocytes (Pani et al., 2006) and inhibition of SERCA2 with thapsigargin (Tg), an inhibitor of SERCA, disrupts a variety of biological processes, particularly during terminal differentiation of keratinocytes (Lowry et al., 1996). Moreover, the loss of one copy of the Atp2a2 allele induced the formation of SCCs in SERCA2 heterozygote (SERCA2þ/) mice (Liu et al., 2001). SERCA2þ/ mice show enhanced tumor susceptibility that is followed by tumor initiation and progression via elevated expression of wild type H-ras, K-ras, and p53 in SERCA2þ/ (Prasad et al., 2005), which suggests that aberrant Ca2þ signaling due to SERCA2 haploinsufficiency is associated with susceptibility to carcinogenesis. SERCA2þ/ mice serve as an animal model for skin tumors (Liu et al., 2001; Prasad et al., 2005). However, in SERCA2þ/ mice keratinocytes, the event of Ca2þ signaling and its related tumor induction and keratinocyte differentiation have not been studied and the role of intracellular Ca2þ in the overall cancerrelated signaling pathway is also uncontested in the primary cultured keratinocyte. In the present work, we investigated the expression of tumorigenic factors in primary cultured keratinocytes as well as the effect of altered SERCA2 expression on Ca2þ signaling.
2.1. Materials Trypsin, ATP, DNase, Hanks' balanced salt solution (HBSS), trypsin, type I collagen, and collagen were purchased from Sigma; Defined keratinocyte media, gentamycin, penicillin, streptomysin, thapsigargin, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA); fura-2 acetoxymethyl ester (fura-2/AM) was purchased from Teflabs (Austin, TX). All other chemicals were used reagent grade. 2.2. Primary keratinocyte culture Wild type (WT) and SERCA2þ/ mice in a Black Swiss background were kindly gifts from Dr. Gary E Shull (University of Cincinnati College of Medicine, USA) (Periasamy et al., 1999), and housed with free access to food and water in a temperaturecontrolled room (23 1 C) under artificial illumination (lights on 06:00 he18:00 h) and 55% relative humidity. All animal protocols were performed according to institutional guidelines of Yonsei University College of Dentistry. Genotypes were determined by PCR analysis of tail DNA as described previously (Periasamy et al., 1999). Dorsal and ventral skin of one mouse was used for keratinocytes culture. The skins were carefully shaved off all body hair, stretched, tissue debris was removed carefully with a scrapper, and the skins were floated on 0.5% trypsin in HBSS for 25 min at 37 C. The floated epidermis was carefully separated from the dermis, neutralized by HBSS including 0.05% DNase and 20% fetal bovine serum, and minced with scissors. The suspension was filtrated through autoclaved nylon nets to take off the remaining body hair and tissue fragments. After sedimentation, the epidermal cells were washed with HBSS, centrifuged for 5 min at 1000 rpm, and re-suspended gently in defined keratinocyte media including 5 mg/ml gentamycin. The cells were cultured on type I collagen-coated dishes in an incubator at 5% CO2 and 37 C and used at 80% confluency. The media was periodically changed every 2e3 days. 2.3. Histology Mouse skins from the lip and genitalia were fixed in 4% paraformaldehyde in phosphate-buffed saline overnight at 4 C, and embedded in paraffin wax. Images of 6 mm thick paraffin sections stained with hematoxylin and eosin (H & E) were obtained using a Leica microscope (Germany). 2.4. Intracellular concentration of Ca2þ ([Ca2þ]i) measurement [Ca2þ]i was determined in primary mouse keratinocytes by fura2/AM in an extracellular physiologic salt solution (PSS), the composition of which was as follows (mM): 140 NaCl, 5 KCl, 5 mM; 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose, titrated to pH 7.4 with NaOH. The osmolarity of the PSS was 310 mOsm. Ca2þ-free medium contained 1 mM EDTA and 1 mM ethyleneglycol-bis-(b-aminoethylether)-N,N,N0 ,N0 -tetra acetic acid (EGTA) in PSS. The primary keratinocytes were cultured on collagen-coated cover glasses for
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measuring [Ca2þ]i. The cells were loaded with 3 mM fura-2/AM for 1 h and after washing with standard solution, [Ca2þ]i was measured by alternately illuminating the cells at wavelengths 340 and 380 nm, and the emitted light was passed through a 510 nm cutoff filter and was collected with a CCD camera and analyzed with a MetaFluor system (Universal Imaging Co., Downingtown, PA). The 340/380 fura-2 ratio was taken as a measure of [Ca2þ]i and fluorescence images were obtained at 3 s intervals. 2.5. Immunoblotting Cells were lysed by adding RIPA (radio-immuno precipitation assay) buffer containing in mM; 10 TriseHCl (pH 7.8), 150 NaCl, 1 EDTA, 1% NP-40, 10 Na3VO4, 10 NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 10 mg/ml PMSF. Lysates were centrifuged at 11,000 rpm for 10 min, and supernatants were collected for immunoblotting. Protein concentration was determined using the BCA assay kit (Pierce, IL). Equal protein amounts (40 mg) were separated by 10% SDS-PAGE gel (Bio-Rad, CA) and electro-transferred onto nitrocellulose membranes. The membranes were then incubated in 5% skim milk powder in TBST (mM); 20 TriseHCl, (pH 7.6), 137 NaCl, and 0.1% Tween 20 for 1 h, and incubated sequentially with primary antibody and followed by horseradish peroxidase-conjugated secondary antibody (SantaCruz, CA). Blotted proteins were visualized by an enhanced chemiluminescence reagent (IntRON, South Korea). 2.6. First-strand cDNA synthesis Total RNA was extracted from cultured keratinocytes and was used for the synthesis of first-strand cDNAs by reverse transcriptase. Reverse transcription was performed for 1.5 h at 42 C in a final reaction volume of 20 ml containing 3 mg of the purified total RNA, 4 ml of 5 reaction buffer (Promega, Madison, WI), 5 ml of dNTPs (each 2 mM), 2 ml of 10 mM dT-ACP1 (50 -CTGTGAATGCTGC GACTACGATIIIIIT-30 ), 0.5 ml of RNasinÒ RNase Inhibitor (Promega), and 1 ml of Moloney murine leukemia virus reverse transcriptase (Promega). First-strand cDNAs were diluted by the addition of 80 ml of purified water for RT-PCR.
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normalized b2M in WT and SERCA2þ/ samples. The data were analyzed with the Thermal Cycler DiceÔ Real Time System analysis software (Takara). The primer sequences of genes are as follows (forward/reverse): tumor-associated calcium signal transducer 1 (TACSTD1, 50 -TTGTGGTGGTGTCATTAGCAGTCA-30 /50 -CACTCAGCAC GGCTAGGCATTA-30 ), crystalline aB (50 -TGATTGAGGTCCACGGCAA G-30 /50 -ACAGTGAGGACTCCATCAGATGACA-30 ), procollagen type XVIII a1 (50 -GTGCCCATCGTCAACCTGAA-30 /50 -AGTTGACCCTGGGA GCCAGA-30 ), nuclear factor I-B (NFI-B, 50 -AATACCTGGAGTCGCG CACA-30 /50 -GGATAGCTTGCGTCGGAAATG-30 ), involucrin (50 -GCAA GACATGCTAGTACCACAGGAG-30 /50 -CTGCTGACCCAGATGCAGTTC-30 ), and b-2 microglobulin (b2M, 50 -GGGAAGCCGAACATACTGAA-30 /50 TCACATGTCTCGATCCCAGT-30 ). The data were expressed as mean SD. The mean expression levels were compared between WT and SERCA2þ/ mice group using ANOVA (SPSS). 2.9. Statistics All data were given as mean SE. Statistical significances of between groups were determined using the Student's t-test. 3. Results 3.1. Squamous cell carcinomas were induced in perineal skin of SERCA2þ/ mice Cancer, which was first observed in 22-week-old SERCA2þ/ mice, reached 100% incidence by the time the mice were 52-week of age. Skin samples were removed from whole body of 52-weekold SERCA2þ/ mice and analyzed by H & E staining. Microscopic view showed the skin lesion was composed of tumor cell nests (T) invading the connective tissues in SERCA2þ/ mice (Fig. 1A). Highpower view of the lesion exhibit common features of cellular atypia with pleomorphism, hyperchromatism, and aberrant accumulations of keratin (arrow) in SERCA2þ/ mice (Fig. 1B). We hypothesized that modulation of Ca2þ signal involved in partial depletion of SERCA2 gene was occurred and which types of genes were involved in SERCA2þ/ keratinocytes. To perform primary keratinocytes culture in subsequent experiments, we used these skin lesions.
2.7. ACP-based GeneFishingÔ PCR for second strand synthesis and direct sequencing
3.2. Altered Ca2þ signaling in SERCA2þ/ keratinocytes
Differentially expressed genes (DEGs) were screened by ACPbased PCR method (Kim et al., 2004) using the GeneFishingÔ DEG kits (Seegene, South Korea). The PCR for second strand synthesis was performed according to the manufacturer's protocol. The amplified PCR products were separated in 2% agarose gel stained with ethidium bromide. Expression levels of DEGs were calculated by the MetaMorph system (Universal imaging Co., Downingtown, PA). The bands of the DEGs were re-amplified and extracted from the gel using the GENCLEANÒII Kit (Q-BIO gene, CA), and directly sequenced with ABI PRISMÒ3100-AvantGenetic Analyzer (Applied Biosystems, CA).
Fig. 2A and B shows that the level of SERCA2b protein was 31.2 1.1% lower in SERCA2þ/ than in WT mice (n ¼ 4). All analyses were conducted on mice that were more than 6 weeks of age to ensure that the epidermal layer was sufficiently mature. Densitometric analysis of the results revealed that the level of PMCA protein was 4.54 0.89 fold higher in SERCA2þ/ than in WT keratinocytes (Fig. 2A and B, n ¼ 4). The Ca2þ signal of keratinocytes obtained from the two mice strains was then evaluated by measuring [Ca2þ]i. The agonistevoked Ca2þ signal was triggered by stimulation of the native P2Y2 receptors with high concentration of ATP (Fig. 2C, n ¼ 10). The ATPinduced maximum increase of [Ca2þ]i of cells maintained in media containing 1 mM extracellular Ca2þ was reduced by 79.4 2.1% in SERCA2þ/ keratinocytes, as reflected in the reduction in the 340/ 380 ratio (Fig. 2E, left panel). Store-operated Ca2þ influx (SOC) was assayed by passive depletion of the stores by inhibition of the SERCA pumps with thapsigargin (Tg) and incubating the cells in nominally Ca2þ-free medium (Fig. 2D, n ¼ 5) for 15 min. Tg-triggered Ca2þ release was lower by 55.1 0.7% in SERCA2þ/ than in WT keratinocytes. SOC activity was 1.58 0.32 fold higher in SERCA2þ/ keratinocytes than in WT keratinocytes (Fig. 2E, right panel).
2.8. Quantitative real-time RT-PCR (Q-rtPCR) Total RNA was isolated the primary cultured keratinocyte cells using Trizol (Invitrogen) reagent. cDNA synthesis was performed using M-MLV reverse transcriptase for reverse transcription (Invitrogen). Quantitative real-time PCR was performed with SYBR premix EX Taq polymerase (Takara) using Thermal Cycler DiceÔ Real Time System (TP800, Takara). The amplification condition was 45 cycles of denaturation at 95 C for 5 s, annealing at 55 C for 20 s, and extension step at 72 C for 20 s. The results of Q-rtPCR were
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Fig. 1. Squamous cell carcinomas were induced in perineal skin of SERCA2þ/ mice. A. The skin lesion of perineum was composed of tumor cell nests (T) invading the connective tissues (H & E, original magnification 40), Indicated scale bar: 1000 mm B. High-power view of the lesion with pleomorphism, hyperchromatism, and aberrant accumulations of keratin (arrow) (H & E, original magnification 200), Indicated scale bar: 100 mm.
Fig. 2. Expressions of Ca2þ signaling proteins and Ca2þ signaling in primary keratinocytes from wild type and SERCA2þ/ mice. A and B, show protein (40 mg) levels of SERCA2b, PMCA, and b-actin in primary keratinocytes. b-actin was used for immunoblotting control. C, Primary keratinocytes obtained from wild type (WT, solid trace) and SERCA2þ/ (dotted trace) mice were stimulated with 100 mM ATP in PSS. D, Cells were perfused with Ca2þ-free medium (open bar) and then treated with 1 mM thapsigargin (Tg) in Ca2þ-free medium for 15 min. After completion of store depletion Ca2þ influx was assayed by the addition of 1 mM Ca2þ to the perfusion media (marked by dark bar). E, Maximum increase in [Ca2þ]i as revealed by the increase in Fura-2 (Rmax-R0) ratio in response to ATP stimulation (left panel). The rate of Ca2þ entry obtained from the period of Ca2þ addition to cells with depleted stores, as in panel D (right panel). Results are depicted as mean S.E.
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Fig. 3. Effect of disrupted Ca2þ signaling on expression of cancer-related signaling proteins. A, show protein level of phospho-ERK (P-ERK), total-ERK (T-ERK), phospho-JNK (P-JNK), total-JNK (T-JNK), phospho-PLCg1 (P-PLCg1), total-PLCg1 (T-PLCg1), NFATc1, and b-actin in primary cultured keratinocytes. B shows the summary of multiple experiments with n 4.
Fig. 4. Patterns of differentially expressed genes (DEGs) in response to SERCA2 haploinsufficiency. A, Relative expression level of ACP-based differentially expressed genes. B, Relative fluorescence unit of 5 genes in 42-week-old mice (n ¼ 4, pairs) using Quantitative real-time PCR. b2M, housekeeping gene, was used mRNA loading control. C, mRNA levels of cytokine-related DEGs. P; positive control, 1; WT, 2; SERCA2þ/, Housekeeping genes (SDHA, HPRT1, and b2M) were used as mRNA controls.
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3.3. Patterns of cancer-related signaling proteins
4. Discussion
Alteration of Ca2þ signaling in Fig. 2 can affect most of cancerrelated signaling pathways. Therefore, we hypothesized in SERCA2þ/ keratinocytes that the activities of caner-related molecules might be changed. The activities of ERK, JNK, phospholipase Cg1 (PLCg1), and NFATc1 in SERCA2þ/ keratinocytes were measured. The steady-state level of the expression of phospho-JNK protein and phospho-ERK protein was increased by 1.37 0.04 and 2.05 0.47 fold in SERCA2þ/ keratinocytes, respectively (n ¼ 4). The level of phospho-PLCg1 protein was 3.06 0.58 fold higher in SERCA2þ/ than in WT keratinocytes (Fig. 3A and B). In addition, the expression of NFATc1 was 3.24 0.09 fold higher in SERCA2þ/ than in WT keratinocytes.
In the present work we found a critical role of Ca2þ signaling in tumor induction and keratinocyte differentiation. Reduction in SERCA2 activity increased both SOCs-mediated Ca2þ influx and PMCA expression in keratinocytes (Fig. 2A). Similar results of SOCsmediated Ca2þ influx had been reported (Pani et al., 2006) that disruption of ER Ca2þ content is associated with an increase in TRPC1 expression in epidermal skin cells from Darier's disease patients and up-regulation of TRPC1 arguments cell proliferation and restrict apoptosis (Bollimuntha et al., 2005, 2006). For the protective role in epithelial layer, keratinocyte differentiation not proliferation plays a crucial role as a normal skin function. Both induction of differentiation and inhibition of proliferation are controlled by Ca2þ increase. Therefore, higher Ca2þ entry across the plasma membrane is important to determine cell fate such as differentiation, proliferation, tumor induction, etc. (Fig. 2D). Prevarskaya et al. (2007) showed the differential role of TRP channels in Ca2þ signal homeostasis of prostate cancer epithelial cells and their potential role in prostate carcinogenesis. Although the key determinants of Ca2þ homeostasis in skin cancer have been outlined, obviously it is important in cancer-associated growth and cell death regulation. Another change in Ca2þ signaling protein was increased phosphorylation of PLCg1, which is likely to increase cellular excitability to maintain the Ca2þ signaling pathway in a more active state under resting condition (Fig. 3). In addition, the expression of the Ca2þregulated transcription factor NFATc1 was markedly increased in SERCA2þ/ keratinocytes (Fig. 3). MAPKs such as JNK, ERK, and p38 are important regulatory proteins that transduce various extracellular signals into intracellular events (Seger and Krebs, 1995) and these MAPKs are modulated by disruption of ER Ca2þ homeostasis (Liang et al., 2006), and ERK activation is an important downstream effecter mechanism for cellular protection from ER stress (Hung et al., 2003). Therefore, the present results of ERK and JNK activation suggest that dysfunction of Ca2þ homeostasis caused by a partial loss of SERCA2 in SERCA2þ/ karatinocytes is related to events of remodeling of the Ca2þ signal-related protein, such as activation of PLCg1, increased SOCs and increased PMCA that activates cell excitability. The most notable work of present study is up-regulated DEGs and down-regulated DEGs in keratinocytes correspond to a variety of carcinogenesis signals (Table 1A and B). For example, TACSTD is involved in the early stages of human lung adenocarcinogenesis (Shimada et al., 2005). The results of our study revealed that in keratinocytes TACSTD1 might be involved in tumor induction. Collagen type XVIII expression is a useful prognostic marker in patients with non-small cell lung carcinoma (Chang et al., 2004) and NFI-B gene is related to tumor generation via fusion (Gronostajski, 2000). Finally, Crystallin aB has chaperone activities in its functional role of holding or folding multiple proteins that have been denatured simultaneously under stress conditions (Chen et al., 2004; Ohto-Fujita et al., 2007). Although it is unclear if the mechanism of TACSTD, NFI-B, procollagen type XVIII, and crystalline aB are related to SERCA2 haploinsufficiency, these up-regulated genes may affect tumor induction in keratinocytes. Down-regulation of involucrin in SERCA2þ/ keratinocyte is related to the differentiation of keratinocytes (Table 1B). Obviously, abnormal Ca2þ signal by SERCA2 depletion inhibits the mechanism of keratinocyte differentiation. This report suggests that disorder of the SERCA2 pump potentially disrupts to build normal skin structure. The TGFbs are multifunctional cytokines families that play a pivotal role in the maintenance of tissue homeostasis. TGFb1 overexpression in the basal layer of the epidermis and hair follicles causes a severe inflammatory skin disorder and epidermal
3.4. Patterns of differentially expressed genes (DEGs) Increased activities of cancer-related signaling proteins in Fig. 3 are likely to affect the expression of cell cycle and other tumorrelated genes (Schubbert et al., 2007). To search for genes affected by the partial deletion of SERCA2 and its downstream-related pathways, the mRNA pool expressed in 52-week-old WT and SERCA2þ/ keratinocytes (n ¼ 4) was compared using an ACP-based gene fishing PCR method. With this approach we found 5 genes that were differentially expressed between the both types of keratinocytes (Fig. 4A). The DEGs that showed the greatest differences were selected for direct sequencing, and the sequences were then evaluated using BLAST searches of the GeneBank database (Table 1). Genes that were increased in SERCA2þ/ keratinocytes included tumor-associated calcium signal transducer 1 (TACSTD1), crystalline aB, nuclear factor I-B (NFI-B), and procollagen XVIII a1, whereas involucrin was decreased in SERCA2þ/ keratinocytes. To confirm these gene profiles, Q-rtPCR was performed using above 5 target primers in 42-week-old mice (Fig. 4B, n ¼ 4, pairs). Cancer cells are capable of attracting different cell types into the tumor environment through angiogenic factors and cytokines (Schubbert et al., 2007; Shchors and Evan, 2007). Therefore, to determine which cytokines were related to the cutaneous tumor induction by SERCA2 haploinsufficiency, ACP-based cytokine PCR was performed. Fig. 4C shows the expression patterns of cytokines in 52-week-old WT and SERCA2þ/ keratinocytes (n ¼ 4). None of the known housekeeping genes analyzed (SDHA; HPRT1, and b2M) was changed in SERCA2þ/ keratinocytes. The expression of TGFb1 and 3 as well as platelet-derived growth factor b (PDGFb), interleukin (IL)-11, and IL-16 were increased in SERCA2þ/ keratinocytes. These results show that partial depletion of SERCA2 can be a potent inducer of cytokines causing cutaneous tumor.
Table 1 Identification of differentially expressed transcripts in response to SERCA2 haploinsufficiency. Identity A. Up-regulation Crystallin aB Nuclear factor I-B Procollagen type XVIII a1 Tumor-associated calcium signal transducer 1 B. Down-regulation Involucrin
GenBank Acc. No.
Base pairs
Sequence homology
BC BC BC BC
848 2637 5029 1492
100% 100% 100% 100%
1902
99% (369/370)
010768 096542 064817 005618
NM 008412
(477/477) (596/596) (585/585) (575/575)
* The percentage expressions are based on BLAST searches of the GenBank databases. The numbers in brackets show the number of bases (query/subject) that were compared.
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carcinogenesis (Li et al., 2006). IL-11 is a pleiotropic cytokine that exhibits anti-inflammatory properties (Theodore et al., 1996), and the inhibition of allogenic lymphocyte reactivity might be due to the direct action of secreted IL-16 (Fujita et al., 2000). The results of the present study shows that SERCA2 haploinsufficiency and perturbation of Ca2þ signaling are powerful signals that induce abnormal expression of the TGFb1 and IL-11 genes (Fig. 4C). However, we presently do not understand how perturbed Ca2þ signal modulate these genes in keratinocytes. In conclusion, our findings show that haploinsufficiency of SERCA2 results in altered intracellular Ca2þ levels and adaptation of the Ca2þ signaling machinery to activate cell excitability. It subsequently evokes the switch-on mechanism of oncogenes expression and the switch-off mechanism of keratinocyte differentiation. Although the precise mechanisms by which cutaneous tumor induction occurs remain to be elucidated, the results of this study demonstrate that SERCA2 haploinsufficiency is linked to the promotion of these genetic abnormalities and may be crucial to tumor induction, at least in a subset of SCCs. Acknowledgements We thank Dr. Gary E Shull for offering the wild-type and SERCA2þ/ mice in a Black Swiss background. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0001658, 20100000315). References Ahn, W., Lee, M.G., Kim, K.H., Muallem, S., 2003. Multiple effects of SERCA2b mutations associated with Darier's disease. J. Biol. Chem. 278, 20795e20801. Berhane, T., Halliday, G.M., Cooke, B., Barnetson, R.S., 2002. Inflammation is associated with progression of actinic keratoses to squamous cell carcinomas in humans. Br. J. Dermatol. 146, 810e815. Bikle, D.D., Ng, D., Tu, C.L., Oda, Y., Xie, Z., 2001. Calcium- and vitamin D-regulated keratinocyte differentiation. Mol. Cell Endocrinol. 177, 161e171. Bollimuntha, S., Ebadi, M., Singh, B.B., 2006. TRPC1 protects human SH-SY5Y cells against salsolinol-induced cytotoxicity by inhibiting apoptosis. Brain Res. 1099, 141e149. Bollimuntha, S., Singh, B.B., Shavali, S., Sharma, S.K., Ebadi, M., 2005. TRPC1mediated inhibition of 1-methyl-4-phenylpyridinium ion neurotoxicity in human SH-SY5Y neuroblastoma cells. J. Biol. Chem. 280, 2132e2140. Chang, H., Iizasa, T., Shibuya, K., Iyoda, A., Suzuki, M., Moriya, Y., Liu, T.L., Hiwasa, T., Hiroshima, K., Fujisawa, T., 2004. Increased expression of collagen XVIII and its prognostic value in nonsmall cell lung carcinoma. Cancer 100, 1665e1672. Chen, J., He, Q.Y., Yuen, A.P., Chiu, J.F., 2004. Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis. Proteomics 4, 2465e2475. Elias, P.M., Ahn, S.K., Denda, M., Brown, B.E., Crumrine, D., Kimutai, L.K., Komuves, L., Lee, S.H., Feingold, K.R., 2002. Modulations in epidermal calcium regulate the expression of differentiation-specific markers. J. Invest. Dermatol.119, 1128e1136. Foggia, L., Hovnanian, A., 2004. Calcium pump disorders of the skin. Am. J. Med. Genet. C Semin. Med. Genet. 131C, 20e31. Fujita, T., Matsumoto, Y., Hirai, I., Ezoe, K., Saito, T., Yagihashi, A., Torigoe, T., Homma, K., Takahashi, S., Cruikshank, W.W., Jimbow, K., Sato, N., 2000. Immunosuppressive effect on T cell activation by interleukin- 16-cDNA-transfected human squamous cell line. Cell Immunol. 202, 54e60. Gronostajski, R.M., 2000. Roles of the NFI/CTF gene family in transcription and development. Gene 249, 31e45. Gunteski-Hamblin, A.M., Greeb, J., Shull, G.E., 1988. A novel Ca2þ pump expressed in brain, kidney, and stomach is encoded by an alternative transcript of the slowtwitch muscle sarcoplasmic reticulum Ca-ATPase gene. Identification of cDNAs
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