Differential expression of proopiomelanocortin (POMC) transcriptional variants in human skin cells

Neuropeptides 47 (2013) 99–107

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Differential expression of proopiomelanocortin (POMC) transcriptional variants in human skin cells E. Zapletal a, O. Kraus b, B. Cˇupic´ a, J. Gabrilovac a,⇑ a b

- Boškovic´ Institute, Division of Molecular Medicine, Laboratory for Experimental Haematology, Immunology and Oncology, Zagreb, Croatia Ruder University Hospital «Sestre milosrdnice», Department of Urology, Zagreb, Croatia

a r t i c l e

i n f o

Article history: Received 18 January 2012 Accepted 22 October 2012 Available online 4 December 2012 Keywords: POMC Transcriptional variants POMC peptide Skin cells Keratinocytes Fibroblasts Cytokine regulation

a b s t r a c t The aims of this study were to examine content and expression level of proopiomelanocortin (POMC) mRNA variants in human dermal fibroblasts (HDF) as compared to primary keratinocytes and HaCaT cells of keratinocyte origin. Primary fibroblasts and keratinocytes were obtained from normal human foreskin. Full-length and total (i.e. the full-length, truncated and/or alternatively spliced) POMC mRNA in skin cells were determined by qRT-PCR using specific probes. The full-length POMC mRNA in HDF is neither constitutively expressed, nor could be induced by corticotropin releasing hormone (CRH) or cytokines interferon c (IFN-c) and transforming growth factorb1 (TGF-b1). However, the truncated/alternatively spliced POMC mRNA variants are constitutively expressed in HDF and could be moderately increased with CRH and the cytokines. Primary keratinocytes, in addition to truncated/alternatively spliced POMC mRNA variants, also constitutively express fulllength POMC mRNA, both being downregulated during in vitro culturing. Unlike primary keratinocytes, HaCaT cells, express only truncated/alternatively spliced POMC mRNA variants. The level of POMC mRNA expression in HaCaT cells was associated with differentiation stage, being higher in more differentiated cells. Thus, in this study we have shown for the first time that HDF do not express the full-length POMC mRNA, either constitutively or upon activation, opposing to primary keratinocytes which constitutively express the full-length POMC mRNA as a minor variant. Although expressing only truncated/alternatively spliced POMC mRNA variant, HDF express POMC peptide, showing that those transcriptional variants are translatable. Truncated/alternatively spliced POMC mRNA variants, expressed both in HDF and keratinocytes are subjected to regulation, implicating their functionality. Furthermore, the IFN-c-induced upregulation at transcriptional level was associated with increased level of POMC peptide detected in HDF lysates. Thus, data of this study have shown that HDF express only truncated/alternatively spliced POMC mRNA variants, which are probably biologically relevant as they could be translated to POMC peptide, both constitutively and upon activation. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Evidence has accumulated showing that mammalian skin contains an equivalent of hypothalamic-pituitary-adrenal (HPA) axis. The human skin HPA axis equivalent includes secretion of corticotropin releasing hormone (CRH), proopiomelanocortin (POMC), POMC-derived peptides and corticosteroids, as well as expression of the corresponding receptors (Slominski et al., 2000; Zmijewski and Slominski, 2011). - Boškovic´ Institute, Division of Molecular ⇑ Corresponding author. Address: Ruder Medicine, Laboratory for Experimental Haematology, Immunology and Oncology, Bijenicˇka c. 54, HR-10002 Zagreb, P.O. Box 180, Croatia. Tel.: +385 1 45 61 011; fax: +385 1 45 61 010. E-mail address: [email protected] (J. Gabrilovac). 0143-4179/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.npep.2012.10.010

POMC, firstly discovered in anterior pituitary corticotroph cells, is a polypeptide precursor of several biologically active peptides: adrenocorticotropic hormone (ACTH), lipotropins (b- and c-LPH), melanotropins (a-, b- and c-MSH) and endorphins (a-, b- and c-END) (Stevens and White, 2010; Wintzen and Gilchrest, 1996). Gene for POMC has two introns and three exons: exon 1 contains untranslated sequences, exon 2 codes for the signal peptide and the first amino acids of the N-terminal peptide and exon 3 codes for the C-terminal part of the N-terminal peptide and several biologically active peptides. There are two well defined POMC promoters: upstream 50 , so called ‘‘pituitary’’ promoter, and downstream promoter within second intron, with wide tissue distribution activity. The first one is responsible for generating full-length variant of POMC mRNA (1.1–1.3 kb) and the second

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one for shorter variant of 0.8 kb (Newell-Price, 2003; Pecori Giraldi et al., 2011). Outside neuroendocrine tissue POMC is also present in placenta, skin and cells of immune system, but in markedly lower quantity, and in different transcriptional variants. Generally, the full-length mRNA predominates in pituitary, whereas the 50 -truncated mRNA variant (ca. 0.8 kb) lacking exons 1, 2 and part of exon 3 predominates in other tissues (Fig. 1). Although the truncated POMC mRNA contains complete sequence coding POMC-derived peptides, it is assumed to be non-functional since the resulting polypeptides would lack the signal sequence necessary for translocation into endoplasmic reticulum and would lack the first part of the N-terminal peptide needed for engagement in secretory pathway (Bicknell, 2008; Raffin-Sanson et al., 2003; Stevens and White, 2010). Thus, products of transcriptional POMC variants may differ in their intracellular processing and secretion. Detection of POMC transcript in the skin confirmed that POMC is generated and secreted locally and not transported from other tissues (Can et al., 1998; Kono et al., 2001; Suzuki et al., 2002). POMC transcripts were also found in isolated cell populations from skin, i.e. in keratinocytes and fibroblasts (Can et al., 1998; Chakraborty et al., 1999; Schauer et al., 1994; Schiller et al., 2001; Slominski et al., 1995, 2005a; Wintzen et al., 1996). POMC mRNA was mostly detected by RT-PCR with probes targeting exon 3, not distinguishing the full-length from the truncated POMC mRNAs (Chakraborty et al., 1999; Schiller et al., 2001; Slominski et al., 1995, 2005a). A few authors by using primers targeting 2nd and 3rd exon or Northern hybridization detected the fulllength POMC transcript (1.2 and 1.3 kb) in isolated skin keratinocytes (Can et al., 1998; Schauer et al., 1994; Wintzen et al., 1996) and fibroblasts (Teofoli et al., 1997). Besides POMC transcript, POMC-derived peptides have also been demonstrated in keratinocytes and fibroblasts (Rousseau et al., 2007; Schauer et al., 1994; Schiller et al., 2001; Slominski et al., 2005a; Teofoli et al., 1999; Wintzen and Gilchrest, 1996; Wintzen et al., 1996), supporting the idea that those cells, like cells of neuroendocrine tissues, express the full-length POMC transcript. The expression of the POMC transcripts in these cells could be upregulated by ultraviolet irradiation and cytokines (Schauer et al., 1994; Teofoli et al., 1997; Wintzen et al., 1996), suggesting their role in skin pigmentation

Signal Peptide

5′′ Exon 1

N-terminal peptide

Exon 2

and peripheral modulation of stress and inflammatory responses (Arck et al., 2006; Slominski, 2007). Functional analogue of local HPA axis in skin was reported and on cellular level ascribed to melanocytes, keratinocytes and fibroblasts. Indeed, in melanocytes CRH upregulated POMC mRNA and POMC peptide, production of ACTH, and finally secretion of corticosteroids (Slominski et al., 2005b). Similarly, functional analogue of HPA axis was reported in human dermal fibroblasts (HDF) (Slominski et al., 2005a). In contrast, in primary keratinocytes complete functional analogue of skin HPA axis could not be demonstrated (Slominski et al., 2005a). However, Cirillo and Prime (2011) reported that HaCaT cells of keratinocyte origin, can synthesize cortisol in response to ACTH. Elements of HPA axis found in keratinocytes were proposed to have role in differentiation (Chakraborty et al., 1999; Slominski et al., 2005a). In this study we examined content and expression level of POMC mRNA variants in human dermal fibroblasts as compared to primary keratinocytes and HaCaT cells of keratinocyte origin. Both constitutive and the activated expression induced by CRH and cytokines interferon-c (IFN-c) and transforming growth factor-b1 (TGF-b1) in HDF were analysed. In addition, the influence of keratinocytes’ senescence and of their differentiation status on the expression level of POMC mRNA variants has been examined. Finally, presence of POMC peptide in primary skin cells was examined and correlated to their POMC transcriptional variants expression. 2. Materials and methods 2.1. Materials Human IFN-c (cat. No. I3265), TGF-b1 (cat. No. T7039) CRH (cat. No. C3042) were purchased by Sigma. 2.2. Skin cell cultures Primary skin cells (keratinocytes and fibroblasts) were obtained from the foreskin of healthy boys, aged 3–8 years. One fibroblast sample originated from a boy aged 7 days. Foreskin samples were non-inflamed and the children were free of any therapy at least

Precursor of biologicaly active peptides

3′

Exon 3

Full-length POMC mRNA Probe 1

Probe 2

3′

5′

Truncated POMC mRNA Probe 2

5′

3′

Fig. 1. Schematic presentation of full-length and truncated POMC mRNA. The boxes represent coding region of the POMC mRNA and vertical lines represent exon-exon boundaries. On schematic presentations of full-length and truncated POMC mRNA thick line shows position of the probe that detects the full length and thin lines show position of the probe that detects both the full-length and the truncated POMC mRNA (modified presentation from Grauerholz et al., 1998).

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1 month before the surgery. The samples were collected after written informed consent of the parents. The study was approved by the Ethical Committee of the Medical Faculty of the University of Zagreb in accordance with the Declaration of Helsinki. Fibroblasts were grown in Dulbecco’s Modified Eagle’s medium with high glucose (DMEM, Sigma, cat. No. D5648) containing 2 mM L-glutamine, 0.1 g/L penicillin and 0.2 g/L streptomycin, and supplemented with 10% foetal bovine serum (FBS, Gibco, cat. No. 10270-106). HDF were used between 3rd and 11th passage. Keratinocytes were isolated according to the procedure described by Boyce and Ham (1983) and cultured in serum-free medium with supplements for their selective growth (DK-SFM, defined keratinocyte serum-free medium; Gibco, cat. No. 10744). Keratinocytes were used between 1st and 4th passages. HaCaT cell line (immortalised keratinocytes, provided by DKFZ, Heidelberg) was grown in DMEM with 10% FBS. All cell cultures were maintained in a humidified atmosphere containing 5% CO2 and the medium was changed every 2 days. Fibroblasts and keratinocytes were detached with 0.25% trypsin– EDTA solution (Gibco, cat. No. 25200) and subcultured. HaCaT cells were detached with 0.05% EDTA (20 min) followed by 0.25% trypsin–EDTA solution (8 min).

and/or alternatively spliced variants we used Custom TaqMan Gene Expression Assay designed on the middle of the coding region of POMC exon 3. Sequences of custom assay were: forward primer 50 -CGACTCCGGGAGGGAGAT-30 , reverse primer 50 -CAGGCTGTGCTCCAGGTC-30 and the probe 50 -CCTGCGCCGTCATC-30 (Fig. 1. The locations of real-time PCR probes for POMC). As a positive control for POMC expression we used cDNA from basophilic anterior pituitary tumour cells (kindly provided by Jelena Knezˇevic´, - Boškovic´ Institute). RPLPO (ribosomal protein, large, PhD, Ruder P0; Hs99999902_m1) and 18S rRNA (eukaryotic 18S rRNA; Hs99999901_s1) were used as endogenous control to normalise the samples. Because our custom assay for POMC could detect genomic DNA we also run no-RT controls (reverse transcription reaction without Reverse Transcriptase) for every sample in order to exclude samples with possible genomic DNA contamination. Reactions were performed using TaqMan Universal PCR Master Mix (Applied Biosystems) in a total volume of 10 ll containing 1 ll cDNA according to the manufacturer’s protocol and the data were collected on an Applied Biosystems 7300 Real-time PCR System. For quantification of POMC mRNA we used comparative Ct method.

2.3. Experimental design

2.6. Detection of POMC peptide by ELISA

Fibroblasts were seeded in 6-well plates (0.5 or 1.0  106 cells per well, depending on treatment duration) in DMEM supplemented with 10% FBS and incubated for 24 h in order to adhere to the surface. Subsequently, the medium was replaced, and the cells treated with CRH, IFN-c and TGF-b1 as indicated in Section 3. The treatment with CRH was done in medium without serum. HaCaT cells were seeded in DMEM with 10% FBS. After reaching semi-confluence the cells were treated with IFN-c or TGF-b1. At the end of treatment the cells were harvested, counted and adjusted to desired concentration for RNA isolation. Primary keratinocytes were seeded at 0.2–0.3  106 cells per 25 cm2 flask and passaged when ca. 80% confluence was reached. In experiments aimed to induce dedifferentiation of HaCaT cell line, the cells were cultured in 25 cm2 flasks in DK-SFM for 6 and 13 days. Medium was replaced every two days. Control cells were cultured in DMEM with 10% FBS. In experiments aimed to collect samples of supernatants and cell lysates for determination of POMC peptide, cells were cultured in 75 cm2 flasks until ca. 80% confluence was reached. Fibroblasts were seeded in DMEM supplemented with 10% of FBS and after their attachment to the surface, the complete medium (10% FCS) was replaced with the medium supplemented with 2% of FBS. Keratinocytes were seeded and cultured in DK-SFM. Medium was replaced one day before cell and supernatant harvest.

Content of POMC peptide was determined in samples of supernatants and cell lysates obtained from fibroblasts and primary keratinocytes. Supernatants were collected in a presence of protease inhibitor phenylmethanesulfonyl fluoride (PMSF, Sigma, cat. No. 78830-1G) and residual cells/cell fragments were precipitated by centrifugation (500g for 10 min). Subsequently the supernatants were concentrated (10 times) by using Vivaspin 2 Centrifugal Concentrators (Membrane: 10,000 MWCO NY, Sartorius Stedim Biotech, product No. VS 02HO1) and ultracentrifugation on 7710g. Samples were aliquoted and stored on 80 °C. Cell lysates were prepared by using cold Radio-Immunoprecipitation Assay (RIPA) buffer that contained protease inhibitor PMSF and 0.01% sodium dodecyl sulphate (SDS). After incubation for 5 min on 4 °C, samples were sonicated (3 times for 10 s), snap frozen in liquid nitrogen and stored on 80 °C. Before ELISA procedure, samples were clarified (centrifugation on 8000g for 10 min) and protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific, cat. No. 23227). Concentration of POMC peptide in samples of supernatants and cell lysates obtained from keratinocytes and fibroblasts were determined by using human POMC ELISA kit (BlueGene, AMS Biotechnology, cat. No. AMS.E01P0133). This ELISA kit applies a quantitative sandwich immunoassay technique and has a sensitivity of 100 pg/ml. Concentration of POMC peptide in samples of cell supernatants obtained from keratinocytes and fibroblasts was additionally examined by using another, more sensitive POMC ELISA kit (USCN, Life Science Inc., cat. No. E91311Hu). That ELISA kit employs the competitive inhibition enzyme technique with minimum detectable concentration of human POMC less than 45.3 pg/ml. For both POMC ELISA kits used no significant crossreactivity or interference between POMC and analogues was declared by the manufacturers. Assay procedures were followed using manufacturer’s instructions with recommended 50 ll of samples. Samples and standards were run in duplicates. Unknown sample concentrations were calculated using standard curve. The data are expressed as pg of POMC per lg of total protein.

2.4. RNA isolation and reverse transcription Total RNA was isolated using High Pure RNA Isolation Kit (Roche, cat. No. 11828665) according to the manufacturer’s protocol. Reverse transcription was performed with 1 lg of total RNA in total volume of 50 ll using TaqMan Reverse Transcription Reagents and random hexamers primers according to the manufacturer’s protocol (Applied Biosystems, cat. No. N808-0234). 2.5. Real-time RT-PCR (qRT-PCR) To measure the content of POMC mRNA we used real-time RT-PCR. For detection of full-length POMC mRNA variant (NCBI Reference Sequence: NM_000939.2) we used TaqMan Gene Expression Assay Hs00174947_m1 whose probe spans exon 1 and exon 2 junction in the 50 untranslated region of the transcript. In order to detect total POMC mRNAs, i.e. full-length, truncated

2.7. Data presentation The POMC mRNA expression was presented either as Ct values or as differences between target and referent genes’ Ct (DCt). Modulation of the POMC transcriptional expression was presented as

E. Zapletal et al. / Neuropeptides 47 (2013) 99–107

3.1. Total POMC mRNA expression in skin cells In this study we firstly intended to reproduce the data of Slominski et al. (2005a), i.e. to detect POMC transcripts in normal human keratinocytes and dermal fibroblasts by using probes, not distinguishing the full-length from the other POMC mRNA variants. To that purpose we used a probe designed on the exon 3 that detects all POMC mRNA variants (in further text designed as total POMC mRNA). Only samples free of genomic DNA were used. Indeed, we detected total POMC mRNA variants, both in HDF and in keratinocytes at approximately comparative level (Table 1). The order of total POMC mRNA expression in samples tested was as follows: pituitary  keratinocytes = fibroblasts > HaCaT. The CRH (106 and 107 M) 30-min and 24-h treatment upregulated the total POMC mRNA in HDF, whereas no change was found after 60-min treatment (Fig. 2A). However, the level of increase was mild (182%, 172% and 163% of the unstimulated control, depending on CRH concentration and treatment duration), opposing the strong increase (24-fold) reported by Slominski et al. (2005a) using the same experimental conditions (60 min treatment with 107 M CRH). 3.2. Full-length POMC mRNA expression in skin cells In order to examine contribution of the full-length POMC mRNA within the total POMC mRNA we used the probe that targets the position between 1st and 2nd exon and detects the full-length POMC mRNA only. Sample of anterior pituitary tumour served as a positive control. Unexpectedly, the data obtained have shown that none of the five fibroblasts’ samples (derived from five different donors) expressed the full-length POMC mRNA, either constitutively or after activation by CRH (data not shown). Even 4-fold increase of template (cDNA) run for 50 cycles did not result in a positive POMC signal (data not shown). In contrast to fibroblasts, all three samples of primary keratinocytes, as well as the sample from basophilic anterior pituitary tumour cells expressed the full-length POMC mRNA (Table 1). The level of expression in keratinocytes was about 400 times lower than in pituitary (difference of about 8 cycles; Table 1). Cells of HaCaT cell line, originating from normal

POMC (fold change)

3. Results

(A)

2.5 none

10-7 M CRH

10-6 M CRH

2 1.5 1 0.5 0 60min

30min

24 hours

Incubation time

(B) 2.5

POMC (fold change)

fold change in respect to the untreated cell sample. All data are expressed as the mean ± standard deviation (s.d.) of triplicates, unless otherwise stated. Number of experiments performed is designated in the Results.

30 min

24 hours

2 1.5 1 0.5 0 0

3

6

IFN-γ (ng/mL)

(C) 2.5

POMC (fold change)

102

30 min

24 hours

2 1.5 1 0.5 0 0

5

10

TGF-β 1 (ng/mL) Fig. 2. CRH, IFN-c and TGF-b1 upregulate expression of total POMC mRNA in HDF. HDF were treated with CRH (A), IFN-c (B) or TGF-b1 (C) of indicated concentrations. Total and full-length POMC mRNA expression was determined after 30-min, 60-min and 24-h treatment with CRH, or after 30-min and 24-h treatment with IFN-c and TGF-b1. No full-length POMC mRNA was detected either constitutively or upon activation of HDF (data not shown). Data of a representative experiment out of three (of two for IFN-c), run in triplicates (expressed as means ± s.d.) are shown.

Table 1 Total and full-length POMC mRNA expression in skin cells and pituitary. Gene expression (Ct) Sample origin

Sample no.

RPLPO

POMCtot

DCt

RPLPO

POMCfull

DCt

Keratinocytes

1 2 3

20.00 20.39 20.36

33.51 34.44 33.63

13.51 14.05 13.28

20.26 20.45 20.19

35.49 35.85 36.51

15.24 15.40 16.32

HaCaT

1 2

20.58 19.40

38.49 36.36

17.91 16.96

20.21 19.74

– –

– –

Fibroblasts

1 2 3 4 5

20.09 20.11 20.98 20.20 20.33

34.22 33.21 35.19 33.25 34.43

14.13 13.10 14.21 13.05 14.10

20.36 20.27 20.21 20.20 20.78

– – – – –

– – – – –

Pituitary

1

24.10

33.74

9.64

24.96

32.06

7.10

Total and full-length POMC mRNA expression in skin cells was presented as Ct and DCt values. A sample from basophilic anterior pituitary tumour cells served as a positive control. RPLPO was used as endogenous control and had very uniform expression. Real-time PCR reactions were run for 50 cycles. POMCtot = total POMC mRNA (i.e. the fulllength, truncated and/or alternatively spliced); POMCfull = full-length POMC mRNA only.

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3.3. Modulation of POMC mRNA expression in skin cells by IFN-c and TGF-b1 One of the proposed roles of local HPA axis in skin is its fine tuning of the local inflammatory/immune responses (Arck et al., 2006). Therefore we examined the effect of the pro-inflammatory cytokine IFN-c and anti-inflammatory TGF-b1 on POMC transcript content and expression level in normal HDF. HDF were treated with IFN-c and TGF-b1 (3 and 6 ng/mL, 5 and 10 ng/mL respectively) for 30 min and for 24 h. Total POMC mRNA (i.e. truncated and/or alternative spliced variants) was detected in control HDF and was moderately upregulated upon treatment with cytokines (maximal increase 184% with IFN-c/Fig. 2B/ and 160% with TGF-b1/ Fig. 2C/, respectively). Lower cytokines’ concentration acted after short (30 min) and higher cytokines’ concentration acted after prolonged (24-h) treatment. No full-length POMC mRNA was detected either in control or in cytokine-treated HDF (data not shown). The ability of IFN-c and TGF-b1 to modulate total and fulllength POMC mRNA expression in HaCaT cell line, originating from normal keratinocytes, was next examined. To that purpose, semiconfluent cultures of HaCaT cells were treated with IFN-c or TGFb1 for 24 h. Subsequently full-length and total POMC mRNA was determined. The treatment of HaCaT cells with either cytokine did not result in a significant change of total POMC mRNA expression (Fig. 3), or in induction of the full-length POMC mRNA (data not shown). Next we wanted to prove our observations by using another housekeeping gene for standardization. To that purpose, 18S rRNA was used along with the previously used RPLPO and the results were compared. It was shown that the expression level of total POMC mRNA in fibroblasts and in keratinocytes was practically the same irrespective of the standard gene used (Table 2). Not only the basal, but also the IFN-c-activated total POMC mRNA

Table 2 Expression level of POMC mRNA in primary skin cells as determined by using two standard genes. Sample FIBRO

FIBRO+IFN-c

KERA

DCt (POMCtot – 18S rRNA) Relative POMCtot mRNA

23.50 1.00

22.64 1.82

23.85 0.78

DCt (POMCtot – RPLPO) Relative POMCtot mRNA

12.45 1.00

11.53 1.88

12.46 0.99

DCt (POMCfull – 18S rRNA) DCt (POMCfull – RPLPO)

– –

– –

25.11 13.72

The level of total POMC mRNA was practically identical in fibroblasts and keratinocytes irrespective of the standard gene used, whereas the full-length POMC mRNA could only be detected in keratinocytes. Real-time PCR reactions were run for 50 cycles. POMCtot = total POMC mRNA; POMCfull = full-length POMC mRNA.

expression was of the same level, regardless of the standard gene used (Table 2.).

3.4. POMC mRNA expression in primary keratinocytes in the course of in vitro culturing Since in vivo expression of POMC mRNA has recently been reported to change with donor age (Pain et al., 2010), we examined whether POMC transcript expression would be changed during in vitro cultivation of primary keratinocytes. To that purpose we followed both the full-length and the total POMC transcripts in keratinocytes obtained from two donors and cultivated from the 1st to the 4th passage. The data obtained have shown that prolonged culturing of keratinocytes was associated with downregulation

1.5

POMC (fold change)

human keratinocytes, did not constitutively express the full-length POMC mRNA (Table 1). Thus, our data have shown that the fulllength POMC mRNA is expressed in normal human keratinocytes, but not in keratinocyte cell line HaCaT and in normal dermal fibroblasts. Since all cell types examined express total POMC mRNA variants, the data suggest that those cells express truncated or alternatively spliced variants of POMC mRNA, and only keratinocytes in addition express the full-length POMC mRNA, as a minor variant (12–42% of the total POMC mRNA).

(A)

Total

Full-length

1

0.5

0 1

2

3

4

Number of passages 1.5

2 1.5 1 0.5 0 None

3

6 IFN-γ

5 10 TGF-β

Treatment (ng/mL) Fig. 3. No significant modulation of total POMC mRNA expression in HaCaT cells by IFN-c and TGF-b1. Semi-confluent HaCaT cells were treated with IFN-c or TGF-b1 of indicated concentrations. Total and full-length POMC mRNA expression was determined after 24-h treatment. No full-length POMC mRNA was detected either constitutively or upon activation of HaCaT (data not shown). Total POMC mRNA expression in HaCaT cells was not significantly affected by IFN-c and TGF-b1. Data of a representative experiment out of two, run in triplicates (expressed as means ± s.d.) are shown.

POMC (fold change)

POMC (fold change)

2.5

(B)

Total

Full-length

1

0.5

0 1

2

3

4

Number of passages Fig. 4. Expression of total and full-length POMC mRNA in primary keratinocytes decreases during in vitro culturing. Primary keratinocytes isolated from two skin samples (A and B) of healthy donors were cultured in vitro for four passages. The cells were seeded at 0.2  106 cells per 25 cm2 and harvested when ca. 80% confluence was reached (6–11 days). Subsequently, expression of POMC mRNA (total and full-length) was determined (run in triplicates). The data (means ± s.d.) are presented as relative changes in respect to the POMC expression detected in the first passage.

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of both full-length and total POMC mRNA variants (Fig. 4), reflecting probably keratinocytes’ senescence.

(A)

DMEM

DK-SFM

3.6. Comparison of transcriptional and peptide POMC expression in primary skin cells Finally, we examined whether the POMC mRNA detected in HDF would result in production of POMC peptide. To that purpose we analysed the content of POMC peptide in fibroblasts and compared it with that in keratinocytes. Both supernatants and cell lysates were analysed by ELISA. The data obtained have shown that no POMC peptide could be detected in supernatants of either cell type (data not shown). Namely, the values obtained in conditioned media were the same as those obtained in control samples of medium alone. That was true for two ELISA kits, with high sensitivity, obtained from two different manufacturers. However, we did found POMC peptide in lysates of HDF, which was 6.5 times lower than that found in lysates of keratinocytes (Fig. 6B). There was no correlation between transcriptional (total POMC mRNA; Fig. 6A) and peptide expression (Fig. 6B) in HDH and primary keratinocytes. Next we examined whether the IFN-c-activated POMC mRNA would also be associated with up-regulation of POMC peptide expression. The data have shown that transcriptional activation was associated with the increased POMC peptide found in HDF lysates (Fig. 6C and D). The data suggest that the truncated/alternatively spliced POMC mRNA found in HDF is translatable, leading to POMC peptide production, which could be increased by IFN-c. 4. Discussion In this study we have examined constitutive and regulated expression of the full-length POMC transcript in two dominant skin populations: epidermal keratinocytes and dermal fibroblasts. The data obtained have shown that primary human keratinocytes express the full-length POMC transcript, whereas primary dermal fibroblasts do not. The latter was true both for constitutive and for activated expression induced by CRH or cytokines IFN-c and

1.5

(B)

p1

1

0.5

0

DMEM

POMC (fold change)

Next we examined the possible influence of differentiation stage on POMC mRNA content and expression level (Chakraborty et al., 1999). To that purpose we used HaCaT cell line of keratinocyte origin. Compared to primary keratinocytes, cells of HaCaT cell line are highly differentiated (Deyrieux and Wilson, 2007) when cultured in conditions of normal calcium concentration in the medium (1.8 mM in DMEM). However, cultured in low-calcium medium HaCaT cells can adopt dedifferentiated phenotype (Deyrieux and Wilson, 2007). Therefore we induced dedifferentiation of HaCaT cells by culturing the cells in low-calcium medium (DK-SFM, Ca++ <0.09 mM) without serum, and examined POMC transcript content and expression level. HaCaT cells cultured in DMEM supplemented with 10% FBS served as control. Indeed, after 6 days (passage 1) of culturing in the low-calcium medium (DKSFM) significant changes in cell morphology and growth were visible (Fig. 5A) and total POMC mRNA variants decreased (i.e. less than 14% of the initial POMC mRNA, Fig. 5B and C). However, expression of the full-length form of POMC mRNA was not detected either in the first or the second passage after dedifferentiation induction of HaCaT cells (data not shown). The data obtained suggest that truncated/alternatively spliced POMC mRNA variants, detected within the total transcriptional variants, are subjected to regulation by dedifferentiation.

POMC (fold change)

3.5. POMC mRNA expression in HaCaT cell line: dependence on differentiation stage

DK-SFM

1.5

(C)

p2

1

0.5

0

DMEM

DK-SFM

Fig. 5. Expression of total POMC mRNA in HaCaT cell line decreases by cell dedifferentiation. HaCaT cells, usually cultured in DMEM with normal Ca++ level (1.8 mM), were transferred to medium with low Ca++ level (DK-SFM; Ca++ < 0.09 mM) in order to induce their dedifferentiation. Expression of POMC mRNA (total and full-length) was determined both in cells grown in DMEM and in DK-SFM after the first (p1; B) and the second (p2; C) passage, i.e. after 6 and 13 days, respectively. No full-length POMC mRNA could be detected in either sample (data not shown). HaCaT cells grown in medium with low Ca++ level showed morphological changes associated with dedifferentiation (A), as compared to HaCaT cells grown in medium with normal Ca++ level. The data (means ± s.d.) of one out two experiments run in triplicates with similar results are shown.

TGF-b1. Since HDF, expressing only truncated/alternatively spliced POMC mRNA variant, also express POMC peptide, the data suggest that those transcriptional variants are translatable. Both keratinocytes and fibroblasts express truncated/alternatively spliced POMC mRNA variants, which are subjected to regulation: downregulation in keratinocytes during in vitro cultivation and dedifferentiation, and upregulation in fibroblasts upon activation with CRH and cytokines IFN-c and TGF-b1. POMC mRNA expression in extra-pituitary tissues and certain brain areas is mirror-like-image of that in pituitary, i.e. only small amount of the full-length POMC mRNA along with the predominant 50 -truncated POMC transcripts are present extra-pituitary (Bicknell, 2008; Grauerholz et al., 1998; Lacaze-Masmonteil et al., 1987; Newell-Price, 2003; Raffin-Sanson et al., 2003). For example, in murine skin, besides dominant truncated, the full-length POMC mRNA was also detected (Slominski et al., 1996). However,

E. Zapletal et al. / Neuropeptides 47 (2013) 99–107

POMC

(A) 7 6 5 4 3 2 1 0

mRNA

(B) Peptide

Total Full-length

Fibro

Kera

Fibro

Kera

Sample origin (C) mRNAtot

(D) Peptide

POMC

3

2

1

0 Fibro

Fibro+IFN

Fibro

Fibro+IFN

Sample origin Fig. 6. Comparison of mRNA and peptide POMC expression in fibroblasts and primary keratinocytes and in IFN-c-activated fibroblasts. Fibroblasts and primary keratinocytes (second passage) were harvested at 80% confluence. The expression of total POMC mRNA and content of POMC peptide in cell lysates (A and B) were determined as described in Section 2. Fibroblasts were seeded in complete medium and after reaching semiconfluence, treated with IFN-c (3.25 ng/ml). 24-h later, the cells were harvested, and the expression of total POMC mRNA and content of POMC peptide in cell lysates (C and D) were determined as described in Materials and Methods. The data are expressed as relative expression in relation to values obtained for untreated fibroblasts (POMC mRNA: DCt POMCtot = 12.45; POMC peptide: 240 pg/lg of total protein). Means ± s.d. of triplicates (POMC mRNA), or duplicates (POMC peptide) are presented.

whereas the truncated POMC mRNA variant was present in the course of the whole hair cycle, the full-length POMC variant appeared only transiently. Using Northern blot Teofoli et al. (1997) reported that HDF from normal skin constitutively express full-length POMC mRNA. However, the data of our study obtained with probes selectively detecting the full-length POMC mRNA variant, could not detect its presence in HDF. In contrast to data of Teofoli et al. (1997), showing no expression of truncated or alternatively spliced POMC mRNA variants in HDF, we have demonstrated their constitutive expression in all samples of HDF tested. Possibility that human skin cells do not express truncated POMC mRNA seems quite unlikely because all other extra-pituitary tissues, in addition to murine skin, express predominantely truncated POMC mRNA variant (Bicknell, 2008; Grauerholz et al., 1998; Lacaze-Masmonteil et al., 1987; Newell-Price, 2003; Raffin-Sanson et al., 2003; Slominski et al., 1996). The apparent discrepancy between data of Teofoli et al. (1997) and data of this study regarding the expression of POMC transcriptional variants observed in HDF is not clear yet. It is very unlikely that sensitivity of the method we used was insufficient since we were able to detect low amount of the full-length POMC mRNA in primary keratinocytes. Another explanation for our inability to detect the full-length POMC mRNA in HDF would be that these cells express an alternatively spliced POMC variant of

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the full-length POMC mRNA that could not be detected with our probe for full-length POMC mRNA designed to span 1st and 2nd exon boundary. Namely, the existence of alternative splice variants of POMC mRNA in pituitary was reported by Lacaze-Masmonteil et al. (1987). The ability of HDF to secrete POMC-derived b-END, a-MSH and ACTH (Teofoli et al., 1999), and to express all elements of functional local HPA axis (Slominski et al., 2005a), implicate that HDF express POMC mRNA variants which are translatable and processed to peptides. The data of our study, showing that HDF expressing only truncated/alternatively spliced POMC mRNA variants, also express POMC peptide have shown that those variants may be functional and give biologically relevant peptides. Using the probe that detects total POMC mRNA variants we have shown that CRH (30-min and 24-h treatment) is capable of increasing POMC mRNA in HDF (whereas no change was found after 60-min treatment) and are partly in line with those of Slominski et al. (2005a). Namely, the level of increase in our study was only modest (180%) as compared to that obtained by Slominski et al. (2005a; i.e. 24-fold after 60 min). However, the authors reported only moderate increase of POMC polypeptide and of POMC-derived ACTH (160% and 250%, respectively). Similar level of CRH-induced upregulation (2-fold after 3-h treatment) of POMC 50 promoter activity was observed (Katahira et al., 1998). A proposed role of the local HPA axis in skin was fine modulation of inflammatory and immune responses in skin (Arck et al., 2006; Slominski, 2007). The idea was supported by observations of increased transcriptional and protein expression of CRH and POMC in inflamed as compared to normal skin (in epidermal keratinocytes and melanocytes and dermal infiltrating cells), suggesting that their expression might be stimulated by inflammatory cytokines (Kono et al., 2001). Indeed, cytokines were generally reported to have stimulatory effect on POMC expression in neuroendocrine cells. Namely, stimulatory effects of several cytokines (IL-1b, IL-2, IL-6, TNF-a) on rat POMC 5’ promoter expression in AtT-20 cells (murine corticotroph cells) were found (Katahira et al., 1998). The data of our study obtained with HDF have shown increased POMC mRNA expression induced by short (30 min) and prolonged (24 h) treatment with IFN-c. Our data are partly in line with those of Katahira et al. (1998) showing that both IFN-a and IFN-c (1 nM) exert acute transient mild stimulatory effect (after 4 h) on POMC promoter expression followed by stronger inhibitory effect (after 8 h and later). However, in their study ACTH secretion was increased after treatment with both INF-a and -c. The mechanism of the stimulatory effect of IFN-c may be presence of IFN-stimulated response elements and IFN-c activation site in POMC 5’-promoter sequence (Darnell et al., 1994; Katahira et al., 1998). The data of our study have shown that TGF-b1, similarly to IFN-c, moderately increased POMC mRNA in HDF. In contrast, Bouret et al. (2001) studying regulation of POMC expression in the brain, reported that TGF-b1 decrease POMC mRNA expression for 50% in rat arcuate nucleus. The reason for the observed discrepancy might reflect differences in species and tissue, and/or in predominant POMC mRNA variants affected, i.e. the full-length vs. truncated/ alternatively spliced one (Bouret et al., 2001, vs. this study, respectively). Teofoli et al. (1997) showed that TGF-b2, another cytokine of the same family, decreased POMC mRNA in HDF, but nevertheless selectively increased secretion of the POMC-derived ACTH (Teofoli et al., 1999). The data of this study have shown that in contrast to HDF, in HaCaT cells originating from normal keratinocytes, expression of total POMC mRNA was not significantly affected by either IFN-c or TGF-b1. Collectively, our data may suggest differential, cell-type-dependent, POMC regulation by cytokines. The data of this study have shown that both full-length and total POMC mRNA expression decreased in primary keratinocytes with prolonged in vitro culturing. Our findings are in line with

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the data reported by Rees et al. (2002) showing that cells of human corticotroph adenoma in late passages lose their ability to express the full-length POMC variant and to secrete ACTH. Namely, prolonged culturing of corticotroph cells was associated with loss of expression of exons 1 and 2, retaining exon 3 only. In contrast to primary keratinocytes, in HaCaT cells of keratinocyte origin, we could not detect any full-length POMC mRNA. Lack of the fulllength POMC mRNA in HaCaT cell line might be due to loss of expression of exons 1 and 2 due to prolonged culturing, as reported by Rees et al. (2002) since those cells derived from a long-term primary culture of human skin keratinocytes (Boukamp et al., 1988). Alternatively, the decreased expression of POMC mRNA with prolonged in vitro culturing of primary keratinocytes observed in our study might be due to cell senescence. Namely, in our study we observed some morphological changes (increased cell volume and change of cell shape) with increased passage of the primary keratinocyte, which we believe may reflect keratinocytes’ senescence. In favour of this interpretation is also our observation that along with increase of the passage number, more time was needed for keratinocytes to reach semiconfluence (i.e. from 5–7 days at early passage, up to 14 days at the 4th passage), thus indicating gradual proliferative senescence. That observation is in line with the reported association of proliferative senescence with keratinocyte ageing in vitro (Norsgaard et al., 1996). Pain et al. (2010) have recently shown that POMC transcriptional expression in human skin keratinocytes increased with age of the donors. However, that was true only for POMC expression in basal keratinocytes, whereas the age-related POMC expression was not observed when whole epidermis was examined. The latter suggest that influence of age on POMC mRNA expression may reflect presence of other keratinocyte differentiation-stages as well as of other cell types also expressing POMC, like melanocytes. Differentiation-associated expression of POMC and of POMCderived peptides has been suggested by several authors. The data presented in this study are in line with that idea, showing that cells of HaCaT cell line downregulate POMC mRNA expression upon dedifferentiation induced by culturing in medium with low Ca++ concentration. Deyrieux and Wilson (2007) described in vitro culture conditions (low calcium medium; i.e. 0.03 mM Ca++) developed to use HaCaT cell line, as a model to study differentiation, which resulted in changes of cells morphology (72 h) and in expression of markers of dedifferentiated basal-like cells (3 weeks). The changes of cells morphology and downregulation of POMC mRNA in HaCaT cells in our study was observed in the first passage (6 days), and sustained in the second one (13 days) at the same level (i.e. less than 14% of the initial POMC mRNA). Our results of downregulation of POMC mRNA expression upon dedifferentiation are in agreement with data of Slominski et al. (1999) who showed that POMC derived a-MSH was strongly expressed in differentiated cells of suprabasal layers of epidermis, whereas only weakly expressed in basal layer and stratum corneum. Similar was shown for b-END immunoreactivity (Kauser et al., 2003, 2004). Chakraborty et al. (1999) also reported more intense staining of ACTH and a-MSH in more differentiated cells of epidermis and an increase in POMC mRNA expression in primary keratinocytes upon in vitro differentiation induced by UVB irradiation. Interestingly, the observed stronger staining in suprabasal layers the authors explained by differentiation-induced enhanced expression of melanocortin-1 receptors, which stronger bound the POMC-derived peptides. Also, expression of POMC processing prohormone convertases (PC1 and PC2), enzymatic machinery that generates POMC-derived peptides, were shown to correlate with increasing differentiation status of in vitro cultured epidermal keratinocytes (Rousseau et al., 2007). The authors observed that expression of PC1, PC2 and regulatory protein 7B2 was higher in keratinocytes with more differentiated phenotype, compared with

those with basal cell phenotype. Thus, as cells differentiate they express more POMC processing enzymes that could efficiently generate POMC-derived peptides. However, Schiller et al. (2001) examining whole human skin reported no difference in PC1 and PC2 staining between differentiated and undifferentiated cells in epidermis. Collectively, decrease of POMC mRNA in cultured primary keratinocytes and in keratinocyte cell line HaCaT, observed in this study, may be due to cell senescence and differentiation stage, respectively. Our data have shown that truncated/alternatively spliced POMC mRNA could be translated, resulting in POMC peptide, found in HDF lysate. In addition, IFN-c-induced up-regulation at transcriptional level was associated with increased level of POMC peptide detected in HDF lysates. The data suggest that truncated/alternatively spliced POMC mRNA could be translated to POMC peptide in a biologically relevant fashion. However, the finding that keratinocytes, expressing the same level of total POMC mRNA as HDF, give rise to 6.5 times more POMC peptide, suggest that a major source of POMC peptide, at least in that type of cells, would be the full-length POMC mRNA variant. 5. Conclusions In summary, by using qRT-PCR and the probe selectively detecting the full-length POMC mRNA, we detected the full-length POMC mRNA only in primary keratinocytes, but not in keratinocyte cell line HaCaT, or in primary dermal fibroblasts. All cell types examined, however, expressed the truncated/alternatively spliced POMC mRNA variants that are subjected to regulation thus implicating their functionality. Demonstration of POMC peptide in lysates of HDF which express only truncated/alternatively spliced POMC mRNA variant has shown that those variants may also be translatable. Their biological relevance is suggested by the observed correlation between transcriptional and POMC peptide up-regulation induced by IFN-c in HDF. Thus, data of this study have shown that HDF express only truncated/alternatively spliced POMC mRNA variants, which are probably biologically relevant as they could be translated to POMC peptide, both constitutively and upon cytokine activation. Conflict of Interest statement The authors declare no conflict of interest. Acknowledgements This work was financially supported by the Croatian Ministry of Science, Education and Sport (Project No. 098-0982464-2520). The - Boškoauthors wish to thank Andrea Ambriovic´-Ristov, PhD, Ruder vic´ Institute, for critical reading of the manuscript. References Arck, P.C., Slominski, A., Theoharides, T.C., Peters, E.M.J., Paus, R., 2006. Neuroimmunology of stress: skin takes center stage. J. Invest. Dermatol. 126, 1697–1704. Bicknell, A.B., 2008. The tissue-specific processing of pro-opiomelanocortin. J. Neuroendocrinol. 20, 692–699. Boukamp, P., Petrussevska, R.T., Breitkreutz, D., Hornung, J., Markham, A., Fusenig, N.E., 1988. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell. Biol. 106, 761–771. Bouret, S., van Chuoi-Mariot, M.T., Prevot, V., Croix, D., Takumi, T., Jegou, S., Vaudry, H., Beauvillain, J.-C., Mitchell, V., 2001. Evidence that TGFb may directly modulate POMC mRNA expression in the female rat arcuate nucleus. Endocrinology 142, 4055–4065. Boyce, S.T., Ham, R.G., 1983. Calcium-regulated differentiation of normal human epidermal-keratinocytes in chemically defined clonal culture and serum free serial culture. J. Invest. Dermatol. 81, 33–40.

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