Bisindolylmaleimide I | Epigenetics Signal

Regulation of 25-hydroxyvitamin D-1-hydroxylase and 24-hydroxylase in keratinocytes by PTH and FGF23

Wenlin Wu1, Hong Fan2, Yi Jiang3, Liyan Liao3, Lusha Li1, Juan Zhao1, Huiling Zhang1, Chandrama Shrestha1, Zhongjian Xie1*

Abstract

Renal 25-hydroxyvitamin D-1α-hydroxylase (1αOHase, CYP27B1) and 24-hydroxylase (24OHase, CYP24A1) are tightly regulated. However, little is known about the regulation of 1α(OH)ase and 24(OH)ase in extra-renal tissue such as the epidermis. The present study was to determine the roles of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF 23) in the regulation of 1α(OH)ase and 24(OH)ase in epidermal keratinocytes as well as epidermal keratinocyte proliferation and differentiation. The results showed that PTH increased the protein level of 1α(OH)ase in human epidermal keratinocyte cell line HaCaT, but had no effect on the level of 24(OH)ase. The effect of PTH on 1α(OH)ase was blocked by the PKC inhibitor. Treatment with FGF23 decreased mRNA and protein levels of 1α(OH)ase and increased mRNA and protein levels of 24(OH)ase in HaCaT cells. The effect of FGF23 on 1α(OH)ase and 24(OH)ase was blocked by the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) inhibitor. In addition, treatment with PTH enhanced levels of differentiation markers including keratin 1, involucrin, loricrin, and filaggrin but reduced levels of BrdU incorporation in HaCaT cells. These effects were inhibited by the PKC inhibitor. FGF23 enhanced proliferation of HaCaT cells, but reduced levels of early differentiation markers including keratin 1 and involucrin and enhanced levels of the later differentiation markers including loricrin and filaggrin. These results suggest that PTH stimulates 1α(OH)ase expression and differentiation of HaCaT cells and inhibits proliferation via PKC. The data also suggest that FGF23 inhibits 1α(OH)ase expression and stimulates 24(OH)ase expression via MAPK/ERK. In addition, FGF23 enhances proliferation and late differentiation and inhibits early differentiation of HaCaT keratinocytes.

KEYWORDS
Keratinocytes; 1,25(OH)2D; PTH; FGF23; Proliferation

INTRODUCTION

Chronic kidney disease (CKD) is a worldwide public health problem (1). The CKD-mineral and bone disorder (CKD-MBD) is closely related cardiovascular events which are likely to be associated with both vitamin D deficiency and reduced circulating levels of 1,25-dihydroxyvitamin D [1,25(OH)2D]. 1,25(OH)2D is the active form of vitamin D. 25-hydroxyvitamin D-1α-hydroxylase [1α(OH)ase] in the kidney is the major enzyme contributing to the synthesis of1,25(OH)2D in the circulation. Converting 1,25(OH)2D to 1,24,25(OH)3D by 25-hydroxylase [24(OH)ase] could significantly reduce the intracellular concentration of 1,25(OH)2D. 24(OH)ase is expressed not only in renal proximal tubular cells but also in keratinocytes (2-5). The expression levels of 24(OH)ase variable in different cell types (5,6), sexes (7), and ages (8,9). The expression levels of 1α(OH)ase altered in some diseases such as certain cancers and psoriasis (5,10,11). The expression of 24(OH)ase is regulated by 1,25(OH)2D in nearly all types of cells targeted by 1,25(OH)2D (12). It has been widely accepted that vitamin D deficiency, loss of kidney mass and inhibition of 1α(OH)ase due to increased secretion of FGF23 contribute to the decrease in circulating levels of 1,25(OH)2D in patients with CKD.
Parathyroid hormone (PTH) is a polypeptide hormone secreted by parathyroid glands. PTH acts through PTH receptor which can be classified into two subtypes including PTH receptor 1 (PTH1R) and PTH receptor 2 (PTH2R). PTH1R is recognized by both PTH and PTH-related peptide (PTHrP) (13). PTH1R is coupled to the Gsα/PKA and the Gq/PLC/PKC(14). Previous studies have suggested that the activation of renal 1,25(OH)2D synthesis by PTH is via activating PKA signaling pathway and the regulation of renal 1,25(OH)2D secretion is via activating PKC signaling pathway (15). PTH stimulates expression of 1α(OH)ase and inhibits expression of 24(OH)ase in proximal tubular epithelial cells and therefore, enhances synthesis of 1,25(OH)2D and suppresses degradation of 1,25(OH)2D in the kidney (16-18).
FGF23 is a protein mainly synthesized by osteocytes and osteoblasts (19) and suppresses renal 1,25(OH)2D synthesis by inhibiting expression of 1α(OH)ase and stimulating expression of 24(OH)ase (20-22). It exerts its activity via FGF receptors including FGFR1/2/3/4 encoded by four distinct genes (23) and requires Klotho for binding to FGFRs (24-26). It is known that FGF23 inhibits renal 1,25(OH)2D synthesis via the MAPK and PI3K/Akt signal pathways (21,22). The increase in FGF23 in response to hyperphosphatemia aggravates the reduction of 1,25(OH)2D in patients with CKD (27-31). However, treatment of bovine parathyroid cells with FGF23 increases 1α(OH)ase mRNA levels (32). In monocytes, 1α(OH)ase and 24(OH)ase are regulated by immunogenic stimuli and cytokines such as γ-interferon (34,35). Treatment with FGF23 in monocytes from healthy donor peripheral blood mononuclear cells and CKD patients’ peritoneal dialysate effluent decreases 1α(OH)ase mRNA levels and synthesis of 1,25(OH)2D (33).
Even though kidney is the major organ for the synthesis of circulating 1,25(OH)2D, many extra-renal tissues also contains 1α(OH)ase and have the ability of converting 25(OH)D to 1,25(OH)2D (36-43). The reduction of the circulating level of 1,25(OH)2D does not seem to be compensated by the extrarenal source of 1,25(OH)2D. However, 1,25(OH)2D synthesis in extra-renal tissues does change in CKD. It has been shown that treatment of anephric patients with supraphysiological concentration of 25(OH)D normalizes their circulating l,25(OH)2D levels (44). Moreover, neutralization of FGF23 with FGF23 antibody significantly elevates serum levels of 1,25(OH)2D in CKD rats (45). These data suggest that extrarenal tissue has the ability of synthesizing 1,25(OH)2D and FGF23 is a major inhibitor of 1,25(OH)2D synthesis at least in kidneys. To date, little is known about the regulation of 25-hydroxyvitamin D 1α(OH)ase and 24(OH)ase in extrarenal tissue. The epidermis contains abundant 1α(OH)ase and was originally used as the source tissue of 1α(OH)ase mRNA for cloning the human 1α(OH)ase cDNA (46). However, little is known about the regulation of 1,25(OH)2D synthesis in the epidermis keratinocytes. Our present study was to investigate the role of PTH and FGF23 in the regulation of 1α(OH)ase and 24(OH)ase expression in epidermal keratinocytes as well as keratinocyte proliferation and differentiation.

MATERIALS AND METHODS

Immunohistochemical staining

Skin, bone, muscle, kidney and liver specimen were isolated from living donors undergoing breast, bone, muscle, kidney or liver operations. Paraffin-embedded 4 mm thick specimens were deparaffinized in turpentine and rehydrated through decreased concentrations of ethanol. Endogenous peroxidase activity was blocked by using 3% H2O2 in methanol for 15 min. The sections were microwaved for 4 minutes with trisodium citrate dihydrate solution (0.125%, pH 6.0) except the sections used for immunohistochemical staining of PTH1R which was incubated with EDTA, and then soaked with phosphate buffered saline (PBS) (pH 7.2-7.4) three times for 5 min. The sections were then pre-incubated with 5% BSA for 30 min to block non-specific bindings. The pretreated slides were incubated overnight at 4°C in a humidified chamber with mouse monoclonal anti-PTH1R antibody (dilution 1:100, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-FGFR1 antibody (dilution 1:100, BOSTER, Wuhan, China), anti-FGFR2 antibody (dilution 1:100, Abgent, San Diego, CA), anti-FGFR3 antibody (dilution 1:100, Abgent, San Diego, CA), anti-FGFR4 antibody (dilution 1:100, Abgent, San Diego, CA), anti-klotho antibody (dilution 1:100, Abcam, St. Louis, MO) and anti-FGF23 antibody (dilution 1:500, Bioss Inc., Boston, MA) or with PBS as the negative control. Following the incubation with these antibodies, the slides were rinsed with PBS three times and were incubated with appropriate biotinylated secondary antibodies for 15 min followed by avidin (ZSGB-BIO, Wuhan, China) and diaminobenzidine (ZSGB-BIO, Wuhan, China). Hematoxylin was used as counter-staining.

Cell culture and treatments

HaCaT cells derived from human keratinocytes were grown in calcium-free Dulbecco’s Modified Eagle’s Medium (DMEM) (Xu-Tai Biologics, Shanghai, China). HK-2 cells derived from human kidney proximal tubule epithelial cells were grown in DMEM-F12. Pfeiffer cells derived from Human diffuse large B-cell lymphoma cells were grown in RPMI 1640. CCL-13 cells derived from normal human liver tissue were grown in DMEM. HEK-293 cells derived from human embryonic kidney were grown in DMEM with high glucose. 10% FBS (BI, Cromwell, CT), penicillin (50 U/ml) and streptomycin (50 mg/ml) (Sigma-Aldrich, St. Louis, Mo) were added to the medium mentioned above. In all experiments, cells were seeded at a density of 1×105/ml and were cultured in 96-well plates for cell proliferation assays by 5-bromo-2’-deoxyuridine (BrdU) incorporation or in 6-well plates for other experiments. Cells at 60% to 70% confluence were starved for 24 hrs in serum free medium and then treated with hPTH1-34 (0 to 10-8 mol/L or 10-8 mol/L; Sigma-Aldrich, St. Louis, Mo), FGF23 (0 to 100 ng/ml or 100 ng/ml; Fitzgerald industries international, Acton, MA) or vehicle [0.2% Dimethyl sulfoxide (DMSO), Sigma-Aldrich, St. Louis, Mo]. In some experiments, cells were pretreated with PKC inhibitor GF109203X (5 μmol/L, Sigma-Aldrich, St. Louis, Mo), or PKA inhibitor H89 (20 μM, Cell Signaling Technology, Beverly, MA) 1 hour before they were treated with hPTH1-34, or MAPK inhibitor U0126 (10 μmol/L, Cell Signaling Technology, Beverly, MA) 0.5 hour before they were treated with FGF23.

Quantitative real-time PCR

Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA), and 1 μg of RNA was reverse-transcribed with iScript (TaKaRa Bio Inc., Ostu, Japan). A SYBR Green RT-PCR Kit (TaKaRa Bio Inc., Ostu, Japan) was used in the real-time PCR assay according to manufacturer’s recommendation. Primers used in these studies are shown in supplementary reference S1. The ΔΔCt method of relative quantification was used to determine the fold change in target gene expression levels.

Western blotting

Total cell lysates were isolated using the RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) containing complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN) and phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China). Phosphatase inhibitors were used in analyzing phosphorylated protein. The Bicinchoninic Acid Protein Assay Kit (Beyotime biotechnology, Shanghai, China) was used to measure protein concentration of the cell lysates. Equal amounts of boiled protein (100 μg protein for PTH1R detection and 20-40 μg protein for other protein detection) was electrophoresed with reducing SDS-PAGE, and electroblotted onto polyvinylidene fluoride membranes (Immobilon-P, 0.45 μM, Millipore, Billerica, MA). After incubation in blocking buffer [100 mM Tris base, 150 mM NaCl, 5% nonfat milk (5% BSA), and 0.5% Tween 20], the blot was incubated overnight at 4°C with primary antibodies. These antibodies include mouse monoclonal anti-PTH1R (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 and anti-β-actin (Abgent, San Diego, CA) and anti-involucrin (Abcam, St. Louis, MO) antibody at 1:1000, goat polyclonal 24(OH)ase antibody (Abcam, St. Louis, MO) at 1:1000, rabbit polyclonal anti-FGFR1 (BOSTER, Wuhan, China), anti-FGFR2, anti-FGFR3 (Abgent, San Diego, CA), anti-1α(OH)ase (Millipore, Billerica, MA), anti-filaggrin, anti-keratin 1, anti-loricrin (Biolegend, San Diego, CA), anti-AKT, anti-phosphate-AKT, anti-JNK, anti-phosphate-JNK, anti-P38, anti-phosphate-P38, anti-ERK1/2, or anti-phosphate-ERK1/2 (Cell Signaling Technology, Beverly, MA) antibody at 1:1000, anti-FGFR4 antibody (Abgent, San Diego, CA) at 1:500, and anti-klotho antibody (Abcam, St. Louis, MO) at 1:800. The membranes were washed a few times and then incubated for 1 hr with anti-IgG secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences Corp., Buckinghamshire, UK) in the blocking buffer. After another series of washes, bound antibody complexes were visualized using the Supersignal Ultra Chemiluminescent Kit (Thermo Fisher Scientific, Inc., Rockford, IL) and exposed to X-ray films.

BrdU cell proliferation assay

The proliferation of HaCaT cell was determined by measuring BrdU incorporation using a BrdU cell proliferation assay kit (Millipore, Billerica, MA) according to the manufacturer’s instructions.

Statistical analysis

Data were analyzed by student’s t-test or two-way ANOVA using SPSS19.0 when applicable. A probability (p) value of less than 0.05 was considered statistically significant ( p< 0.05).

RESULTS

Expression of PTH1R in normal human epidermis and HaCaT cells

Immunohistochemistry was carried out to examine the presence of PTH1R in normal human epidermis. Human renal tubular epithelial cells (HK-2) served as a positive control. Primary antibody replaced by PBS served as negative control. The results showed that there was a positive staining of PTH1R in basal keratinocytes in normal human epidermis. In contrast, HK-2 cells used as a positive control were positively stained and there was no staining in Pfeiffer cells used as the negative controls (Figure 1A, 1B and 1C). Western blot and qRT-PCR were used to examine the expression of PTH1R in HaCaT cells. The results indicate that the protein and mRNA of PTH1R are present in HaCaT 1-34 enhanced the protein level of 1α(OH)ase, but had no effect on 24(OH)ase in HaCaT cells Treatment with hPTH1-34 (10-12-10-8 mol/L) induced the protein level of 1α(OH)ase in HaCaT cells in a dose-dependent manner, but had no effect on the protein level of 24(OH)ase in HaCaT cells after 24 hrs stimulation (Figure 1D). However, treatment with the same concentration of hPTH1-34 had no effect on mRNA levels of 1α(OH)ase and 24(OH)ase after 24 hrs stimulation (Figure 1E).These results indicate that PTH induces 1α(OH)ase expression but has no effect on 24(OH)ase expression.

PKC inhibitor GF109203X blocked the effect of hPTH1-34 on 1α(OH)ase protein in HaCaT cells

To determine whether PKA or PKC is involved in PTH-induced 1α(OH)ase expression in keratinocytes, HaCaT cells were treated with the PKC or PKA inhibitor and then treated withhPTH1-34 at a concentration of 10-8 mol/L. The results showed that the effect of hPTH1-34 on 1α(OH)ase was blocked by the PKC inhibitor GF109203X at a concentration of 5 μM, but not by the PKA inhibitor H89 at a concentration of 20 μM (Figure 1F). These results suggest that PTH induces 1α(OH)ase expression via PKC.

Effects of hPTH1-34 on proliferation and differentiation of HaCaT cells

To determine the effect of PTH on keratinocyte proliferation and differentiation, HaCaT cells were treated with hPTH1-34 at concentrations between 10-10 to 10-8 M. The results showed that treatment with hPTH1-34 reduced proliferation and induced differentiation of HaCaT cells at a dose-dependent manner (p<0.05) (Figure 2 A-C), suggesting that PTH suppresses keratinocyte proliferation and induces keratinocyte differentiation.

Effects of hPTH1-34 on proliferation and differentiation of HaCaT cells were blocked by the PKC inhibitor

To determine whether the effect of hPTH1-34 on the proliferation and differentiation of keratinocytes is mediated by PKC or PKA. HaCaT cells were treated with PKC inhibitor GF109203X at a concentration of 5 μM or PKA inhibitor H89 at a concentration of 20 μM and then treated with hPTH1-34 at a concentration of 10-8 mol/L and cell proliferation was examined. The results showed that hPTH1-34 reduced proliferation and induced differentiation of HaCaT cells and these effects were abolished by the PKC inhibitor, but not the PKA inhibitor (p<0.05) (Figure 2 D-F). These results suggest that PTH suppresses keratinocyte proliferation and induces keratinocyte differentiation via PKC but independent of PKA.

FGFR2/3/4 and Klotho, but not FGFR1 and FGF23, are expressed in normal human epidermis and HaCaT cells

Immunohistochemistry was carried out to examine the presence of FGFR1/2/3/4, FGF23 and klotho in normal human epidermis. The human renal tubular epithelial tissue was used as a positive control for FGFR1/3/4. The human liver tissue was served as a positive control for FGFR2. The human distal renal tubular epithelial tissue was used as a positive control for klotho. The human bone tissue was used as a positive control and the human muscle tissue served as a negative control for FGF23. The negative control was performed by omitting the primary antibody. The results showed that renal tubular epithelial cells were positively stained for FGFR1/3/4 (Figure 3A) and liver cells were positively stained for FGFR2 (Figure 3B). Osteocytes embedded in the bone matrix and osteoblasts located at the surface of trabecular bone were positively stained for FGF23 (Figure 3C). However, there was no immunostaining for FGF23 in muscle cells and in keratinocytes (Figure 3C). Moreover, distal renal tubular epithelial cells were positively stained for klotho (Figure 3F). In the human epidermis, there was a positive staining for FGFR2/3/4 and klotho in keratinocytes, but not for FGFR1 and FGF23 (Figure 3A, 3B, 3C and 3F). There was no staining in the negative control (Figure 3A, 3B and 3F).
Western blotting and qRT-PCR were used to examine the expression of FGFR1-4 and klotho in HaCaT cells. The HK-2 cell line was used as a positive control for FGFR1/3/4. Human liver cell line CCL-13 cell severed as a positive control for FGFR2. HEK-293 cells severed as a positive control for klotho. Pfeiffer cell severed as a negative control for FGFR1/2/3/4. The results showed that HK-2 cells expressed both protein and mRNA of FGFR1/3/4 (Figure 3D and 3E). CCL-13 cells expressed both protein and mRNA of FGFR2 (Figure 3D and 3E). HEK-293 cells expressed both protein and mRNA of klotho (Figure 3G and 3H). HaCaT cells expressed both protein and mRNA of FGFR2/3/4 (but not the FGFR1), and klotho (Figure 3D, 3E, 3G, and 3H). Pfeiffer cells expressed neither protein nor mRNA of FGFR1/2/3/4 and klotho (Figure 3D, 3E, 3G, and 3H).

FGF23 reduced the expression of 1α(OH)ase and enhanced the expression of 24(OH)ase in HaCaT

Treatment of HaCaT keratinocytes with FGF23 (5-100 ng/ml) reduced the protein and mRNA levels of 1α(OH)ase and increased the protein and mRNA levels of 24(OH)ase at 24 hrs (Figure 4A and 4B).

The FGF23 signaling in HaCaT keratinocytes

We examined whether FGF23 activates the MAPK or AKT signaling pathway in HaCaT cells. The results showed that the expression of MAPK phosphate-ERK1/2 and phosphate-Akt was induced by FGF23 (100 ng/ml), but the expression of MAPK phosphate-P38 and MAPK phosphate-JNK was not induced by FGF23 (100 ng/ml) (Figure 4C).

The effect of FGF23 on 1α(OH)ase and 24(OH)ase in HaCaT cells was blocked by the MAPK ERK1/2 inhibitor

Then we investigated whether MAPK-ERK1/2 mediates FGF23-regulated expression of 1α(OH)ase and 24(OH)ase in HaCaT cells. The results showed that treatment with FGF23 (100 ng/ml) decreased the protein and mRNA levels of 1α(OH)ase and increased the protein and mRNA levels of 24(OH)ase after 24 hrs, but the effects of FGF23 were blocked by the MAPK-ERK1/2 inhibitor U0126 (10 μmol/L) in HaCaT cells (Figure 4D and 4E).

Effects of FGF23 on the proliferation and differentiation of HaCaT cells

To examine whether FGF23 regulates proliferation and differentiation of the epidermal keratinocytes, HaCaT cells were treated with FGF23 at concentrations between 5 and 100 ng/ml) and cell proliferation and differentiation were examined. The results showed that FGF23 induced proliferation of HaCaT cells (Figure 4F). The regulation of expression of differentiation of markers was seen only at protein levels but not mRNA levels (Fig 4G and 4H).

DISCUSSION

In the present study, the results show that both the human epidermis and HaCaT cells express PTH1R, FGFR2/3/4and klotho, but not FGFR1. PTH increases the protein level of 1α(OH)ase, but has no effect on 24(OH)ase. The results also suggest that the effect of PTH on 1α(OH)ase is via PKC, but not via PKA. FGF23 reduces mRNA and protein levels of 1α(OH)ase and enhances mRNA and protein levels of 24(OH)ase in HaCaT cells. FGF23 stimulates phosphorylation of MAPK ERK1/2 and AKT. Moreover, the effects of FGF23 on 1α(OH)ase and 24(OH)ase are via MAPK ERK1/2. In addition, PTH increases levels of differentiation markers including keratin 1, involucrin, loricrin, and filaggrin and reduces levels of proliferation marker BrdU in HaCaT cells. These effects are via PKC, but not via PKA. FGF23 enhances proliferation of HaCaT cells, but decreases early differentiation markers, while increases later differentiation markers.
PTH1R is present in some classical PTH target organs such kidney, bone and parathyroid glands and is also expressed in non-classical PTH target cells such as human fibroblasts and rat keratinocytes (47-49). To date, there are no consistent results regarding PTH1R expression in human keratinocytes. Two decades ago, Hanafin and Sharpe et al failed to identify PTH1R in primary cultures of human keratinocytes by Northern blotting, RT-PCR or RT-PCR together with Southern blotting (49,50). However, Orloff et al identified PTH1R expressed in primary human keratinocytes by Northern blotting and the PTH1R expressed in primary human keratinocytes was a new type of PTH1R, because the length of the PTH1R mRNA identified was shorter than the classical PTH1R.
Moreover, Orloff et al found that PTH1R in human keratinocytes was homologous to the classical PTH1R mRNA in the regions of G, M1 and M2 (48). Henderson et al identified PTH1R in immortalized human RHEK-1 keratinocytes by using [125I] PTH1-34 binding assays (51). Recently, Muehleisen et al found that the level of PTH1R in primary human keratinocytes was low when cells were not exposed to 1,25(OH)2D (52). Although there was no consistent result of PTH1R expression in human keratinocytes, it was observed that human keratinocytes responded to PTH (47,48,50,53,54). The results from the present study indicate that keratinocytes in the basal layer of the epidermis expresses PTH1R and 1α(OH)ase. HaCaT keratinocytes also express protein and mRNA of PTH1R although the expression level is lower than HK-2 cells.
It is known that PTH1R is required for the regulation of 1,25(OH)2D synthesis by PTH. Many extra-renal cells such as macrophages do not respond to PTH because of lacking PTH1R (55,56). The expression of PTHR in keratinocytes is a prerequisite to PTH response. Our present result showed that PTH promoted expression of 1α(OH)ase protein in keratinocytes. PTH stimulates expression of 1α(OH)ase in renal proximal tubule cells and HaCaT keratinocytes appear to be via different mechanisms. Our results indicate that the stimulation of 1α(OH)ase by PTH is mediated by PKC in keratinocytes, which is contrary to the stimulation of 1α(OH)ase mediated by PKA. Our results also showed that PTH increased 1α(OH)ase expression in HaCaT cells at protein level but not in mRNA level. In contrast, PTH enhances both protein and mRNA of 1α(OH)ase in the kidney (57-60).
The present studies showed that PTH increased levels of differentiation markers including keratin 1, involucrin, loricrin, and filaggrin and reduced levels of proliferation marker BrdU in HaCaT cells. These effects were blocked by the PKC inhibitor, but not by the PKA inhibitor. Previous studies have shown that PKC is critical for keratinocyte differentiation (61-63). Therefore, PKC appears to mediate both basal and PTH induced differentiation of keratinocytes.
PTHrP is highly homologous to PTH (64-66). HaCaT and primary keratinocytes produce PTHrP (67-69). Knockdown of PTHrP blocks calcium-induced keratinocytes differentiation (67, 68). Thus, PTH and PTHrP are two potential candidates for inducing differentiation of keratinocytes. On the other hand, 1,25(OH)2D promotes keratinocyte differentiation and inhibits keratinocyte proliferation (70-72). When l,25(OH)2D is decreased, PTH would increase to promote the l,25(OH)2D synthesis in
HaCaT keratinocytes. Increased l,25(OH)2D would induce keratinocyte differentiation. Therefore, PTH directly and indirectly induces differentiation of keratinocytes. The indirect induction of keratinocyte differentiation by PTH appears to be via inducing of 1α(OH)ase. 1,25(OH)2D synthesis in the kidney is not only regulated by PTH, but is also regulated by FGF23. The receptors of FGF23 include FGFR1/2/3/4 (19). The binding affinity of FGF23 to FGFRs is very low and it requires its co-receptor klotho to enhance the affinity of FGFR to FGF23 (26). We found both human epidermis and HaCaT cells expressed FGFR2/3/4 and klotho. FGF23 is known to inhibit renal tubule reabsorption of phosphorus by interacting with FGFR1 (31). However, the epidermal keratinocytes do not express FGFR1 which mediates regulation of phosphorus reabsorption in the kidney. Therefore, it remains unclear which receptor mediates the inhibitory effect of FGF23 on renal 1,25(OH)2D synthesis.
We have found that FGF23 decreases mRNA and protein levels of 1α(OH)ase and increases mRNA and protein levels of 24(OH)ase in HaCaT cells. Moreover, the effects of FGF23 on 1α(OH)ase and 24(OH)ase are via MAPK ERK1/2. Thus, regulation of 1,25(OH)2D synthesis by FGF23 inHaCaT cells appears to be similar to renal tubular cells (20-22).
In the present study, we have found FGF23 stimulates phosphorylation of MAPK ERK1/2 and AKT in HaCaT cells. The effects of FGF23 on 1α(OH)ase and 24(OH)ase in HaCaT cells were blocked by the MAPK ERK1/2 inhibitor U0126, suggesting that ERK1/2 mediates the effect of FGF23 on the expression of 1α(OH)ase and 24(OH)ase. Further research is needed to investigate how ERK1/2 mediates the effect of FGF23 on the expression of 1α(OH)ase and 24(OH)ase. In addition to the regulation of 1α(OH)ase and 24(OH)ase, FGF23 induces proliferation and late differentiation of keratinocytes but reduces early differentiation of keratinocytes. The significance of regulation of proliferation and differentiation of keratinocytes by FGF23 is not clear yet.
Previous studies have demonstrated that treatment of anephric patients with supraphysiological concentration of 25(OH)D normalizes their circulating l,25(OH)2D levels(44), suggesting that extra-renal tissue synthesizes 1,25(OH)2D and may contribute circulating 1,25(OH)2D. The present studies indicate that FGF23 reduces expression 1α(OH)ase and enhances expression 24(OH)ase in HaCaT cells, and therefore might reduce the level of 1,25(OH)2D in these cells. Reduced 1,25(OH)2D may result in increased proliferation and decreased differentiation. This seems to be paralleled to the stimulatory effect of FGF23 on HaCaT cell proliferation and the inhibitory effect of FGF23 on HaCaT cell differentiation.
The decrease in 1α(OH)ase expression and the increase in 24(OH)ase expression in epidermal keratinocytes caused by FGF23 might cause a decrease in 1,25(OH)2D levels in the cell. The present studies also showed that PTH raised the expression of 1α(OH)ase but had no effect on the expression of 24(OH)ase in HaCaT cells, presumably increased the level of 1,25(OH)2D in HaCaT cells. Therefore, maintaining sufficient levels of 25(OH)D and relative high level of PTH and reducing the level of FGF23 may have the possibility of increasing the level of 1,25(OH)2D in the epidermis in patients with CKD.
The present studies have several limitations. The first limitation is that the conclusion was drawn based on in vitro experiments. In vivo experiments are required to determine the regulation of extra-renal production of 1,25(OH)2D by PTH and FGF23. The second limitation is that a keratinocyte cell line was used in the present study. Primary keratinocytes are needed to confirm the results in the future experiments. The third limitation is that the direct measurement of 1,25(OH)2D levels in the cell was not included in the study.
In conclusion, human epidermal keratinocytes express PTH1R, FGFR2/3/4 (but not FGFR1), and klotho. PTH induces expression of 1α(OH)ase and HaCaT keratinocyte differentiation and reduces HaCaT keratinocyte proliferation via PKC, but has no effect on the expression of 24(OH)ase. In contrast, FGF23 reduces expression of 1α(OH)ase and enhances expression of 24(OH)ase via activating ERK1/2. In addition, FGF23 induces proliferation of HaCaT keratinocytes, but reduces early differentiation and enhances later differentiation of these cells.

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