Protein Kinase C Alpha Acting Through Phorbol-12-Myristate 13-Acetate Regulates Nephronectin Gene Expression Via c-Jun and c-Fos Transcription Factors

Nephronectin (Npnt) is an extracellular matrix protein and ligand of integrin α 8 β 1 known to promote differentiation of osteoblasts. A search for factors that regulate Npnt gene expression in osteoblasts revealed that phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), had a strong effect to suppress that expression. Research was then conducted to elucidate the signaling pathway responsible for regulation of Npnt gene expression by PMA in osteoblasts. Treatment of MC3T3-E1 cells with PMA suppressed cell differentiation and Npnt gene expression. Effects were noted at a low concentration of PMA, and were time- and dose-dependent. Furthermore, treatment with the PKC signal inhibitor Gö6983 inhibited down-regulation of Npnt expression, while transfection with small interfering RNA (siRNA) of PKCα, c-Jun, and c-Fos suppressed that down-regulation. The present results suggest regulation of Npnt gene expression via the PKCα and c-Jun/c-Fos pathway.


Introduction
The extracellular matrix surrounding cells is known to be involved in various biological functions, such as cell proliferation, differentiation, and apoptosis 1−3 . Several studies have suggested that the interaction of cells with the extracellular matrix is indispensable for histogenesis and maintenance of biological functions 4,5 . Nephronectin (Npnt) is an extracellular matrix protein considered to play critical roles in the development and function of various tissues 6,7 . Npnt gene expression is seen in calci cation tissues, especially in osteoblasts, thus in order to investigate osteoblast functions, we have performed experiments to elucidate the pattern of Npnt gene expression with several different reagents. In previous studies, we found that 1α,25-dihydroxyvitamin D 3 and Wnt3a promoted Npnt gene expression 8, 9 , whereas TGF-β, TNF-α, IL-1β, OSM, FGF-2, and inorganic phosphate suppressed that expression 10−15 . Those results suggest that Npnt gene expression in osteoblasts is regulated via various factors. In a study conducted by Kahai S et al., an osteoblast-transfected Npnt gene expression vector was shown to promote differentiation 16 . Moreover, that differentiation was strongly promoted in cells in which the expressed region included EGF repeats. Also, in osteoblasts showing a high level of expression of mRNA 3'UTR in the Npnt gene, the calci cation nodule was highly promoted 17 .
PMA is a phorbol ester from the spurge family of plants and the main ingredient in croton oil, which causes strong carcinogenetic promotion activity. Protein kinase C (PKC), which is activated by PMA, is a family of serine-threonine kinases that catalyze various biochemical reactions critical for the function of many cellular components, such as cell differentiation and proliferation 18, 19 . The PKC family consists of 13 isoforms that can be divided into four subgroups based on their activated pattern 20 . Classical PKCs (cPKCs; α, βI, βII, γ) require Ca 2+ /diacylglycerol (DAG)/phosphatidylserine (PS), new PKCs (nPKCs; δ, ε, η, θ) require DAG/PS, and atypical PKCs (aPKCs; λ/ι, ζ) require PS, while so-called PKC-related kinases (PRKs; 1, 2, 3), which are structurally distinct PKCs, require only PS for activation 20 . Activator protein 1 (AP-1) is a dimer consisting of the c-Jun, c-Fos, activating transcription factor (ATF), and musculoaponeurotic brosarcoma (MAF) families 21 . In most cells, the AP-1 morphology is predominantly a Jun/Fos heterodimer, which has a high a nity for binding to the PMA response component, thus is referred to as an AP-1 site 22 . It has also been reported that tumor promoters, such as PMA and epidermal growth factor, induce AP-1 activity 23 . The relationship of PKC and AP-1 has been investigated by analyses of their molecular mechanisms 24,25 .
In the present study, PMA was found to strongly inhibit Npnt gene expression through PKCα and the c-Jun/c-Fos pathway.

Cell culture
The osteoblast-like cell line MC3T3-E1 was maintained in MEMα with L-glutamine and phenol red

Statistical analysis
Values are expressed as the mean ± SD. A two-sided unpaired Student's test was used for statistical analysis. Statistical differences were considered to be signi cant when the p value was < 0.05.
To investigate the effect of PMA on osteoblastic differentiation, MC3T3-E1 cells were cultured with BMP-2 (100 ng/ml) in the absence or presence of PMA (5 nM) for three days. ALP activity in cells cultured with BMP-2 was shown to be increased, whereas it was signi cantly suppressed when cells were cultured in the combination of BMP-2 and PMA (Fig. 1Aa, b). At the same time, the gene expressions of Alp and Osteocalcin, differentiation markers of osteoblasts, were investigated. Both Alp and Osteocalcin gene expressions induced by BMP-2 were suppressed by PMA. These results showed that PMA suppressed BMP-2 induced osteoblast differentiation (Fig. 1B) 26 .
Npnt gene expression is suppressed by PMA in dose and time-dependent manner.
PMA, a phorbol ester, is known to activate the PKC signaling pathway. To determine whether PMA activated the PKC signaling pathway in MC3T3-E1 cells, Marcks phosphorylation was examined, as previous studies have reported that it was phosphorylated by PKC activation 27,28 . Marcks was remarkably phosphorylated by PMA (Fig. 2A). The effect of PMA on Npnt gene expression was also examined and the results showed that expression to be signi cantly down-regulated by PMA (Fig. 2B). Next, the effects of PMA on dose-and time-dependent Npnt gene expression were investigated. That expression was signi cantly decreased by PMA at 3.2 nM and reached a plateau at 32 nM (Fig. 2C), while it was also signi cantly decreased by 10 nM of PMA at 12 hours and then reached a plateau at 24 hours (Fig. 2D). These results suggest that Npnt gene expression is suppressed by PMA in a dose and timedependent manner.
PKCα is involved in down-regulation of Npnt gene expression by PMA.
To verify whether down-regulation of Npnt gene expression by PMA is involved in the PKC signaling pathway, MC3T3-E1 cells were pretreated with Gö6983, known as a broad-spectrum PKC inhibitor, before PMA stimulation. Phosphorylation of Marks by PMA did not occur following pretreatment with Gö6983 ( Fig. 3A), while down-regulation of Npnt gene expression by PMA was inhibited by Gö6983 (Fig. 3B). These results suggest that Npnt gene expression is involved in the PKC signaling pathway.
It has been reported that PKCα is highly expressed in MC3T3-E1 cells 29 . To verify its involvement in down-regulation of Npnt gene expression, MC3T3-E1 cells were pretreated with or without Pkcα siRNA, and thereafter with PMA alone or in combination. When Pkcα siRNA decreased the cellular protein level of Pkcα (Fig. 3C), down-regulation of Npnt gene expression by PMA was inhibited (Fig. 3D). These results indicate that PKCα is involved in down-regulation of Npnt gene expression by PMA.

Both of c-Jun and c-Fos are involved in down-regulation of Npnt gene expression.
It has been reported that regulation of gene expression by PMA is involved in activation of PKCα and thereafter of AP-1 30 . To investigate the involvement of c-Jun and c-Fos as transcription factors, which compose AP-1, on down-regulation of Npnt gene expression, MC3T3-E1 cells were pretreated with or without c-Jun, c-Fos siRNA, and then treated with PMA alone or in combination. When c-Jun siRNA decreased the cellular protein level of c-Jun (Fig. 4A), down-regulation of Npnt gene expression by PMA was inhibited (Fig. 4B), and when c-Fos siRNA decreased the level of c-Fos mRNA (Fig. 4C), downregulation of Npnt gene expression by PMA was inhibited (Fig. 4D). These results demonstrated that the transcription factors c-Jun and c-Fos are involved in down-regulation of Npnt gene expression by PMA.

Discussion
The present ndings indicate that PMA, known to suppress osteoblast differentiation, downregulates Npnt gene expression. That downregulation was shown to be mediated via PKCα, and further via c-Jun and c-Fos, which are transcription factors in PKC signaling. Nakura A. et al., demonstrated that knockdown of PKCα gene expression promoted osteoblast differentiation and their results also suggest that PKCα suppresses osteoblast differentiation 31 . Furthermore, Galea, G.L. et al. reported that PKCα knockout mice, which show a phenotype similar to human Gaucher disease, had bone formation into the medullary space of the femur. Moreover, osteoblasts derived from those mice showed elevated osteoblast differentiation markers, such as Runx2, Osterix, Col1A1, and Osteocalcin 32 . Together, these results suggest that PKCα negatively regulates bone formation. Additionally, the present results indicate that PKCα negatively regulates promotion of osteoblast differentiation, with one of the causes considered to be a decrease in Npnt gene expression due to PKCα, though further studies are required to con rm that association.  34 . That study also noted that the promoter region of the ckβ gene has binding sites for the transcription factors GATA and Ets, and mutations in those binding sites inhibited suppression of the promoter activity of the ckβ gene by PMA. It is considered that these transcription factors may be involved in suppression of Npnt gene expression by PMA, though additional research is needed to verify their relationship.
In conclusion, we found that PKCα suppresses Npnt gene expression via c-Jun and c-Fos transcription factors (Fig. 5  Effects of PMA on BMP-2 induced osteoblast differentiation in MT3T3-E1 cells. (A) (a) MC3T3-E1 cells were treated with or without BMP-2 (100 ng/ml) in the presence or absence of PMA (5 nM) for three days. For quanti cation of ALP activity, cells were disrupted by sonication in 50 mM Tris-HCl containing 0.1% NP40. ALP activity was determined following incubation with the substrate p-nitrophenylphosphate and using absorbance at 405 nm. (b) For ALP staining, cells were xed using 10% formalin in PBS and then ALP activity was visualized using a mixture of 0.1 mg/ml Naphthol As-Mx, 0.6 mg/ml phosphate, and Fast blue BB salt. (B) Total cellular RNA was extracted, then mRNA levels of Alp, Osteocalcin, and Gapdh were examined using quantitative real-time PCR analysis. Results are shown as the mean ± SD of three samples. **P <0.01, Student's t test. were starved for 16 hours in serum-free medium. Next, they were pretreated with or without Gö6983 (500 nM) for one hour, and then with PMA (5 nM) alone or in combination for ve minutes. Proteins were extracted and subjected to western blotting to detect phosphorylation of Marks (p-Marks) and actin. (B) MC3T3-E1 cells were pretreated with or without Gö6983 (500 nM) for one hour, and then treated with PMA (5 nM) alone or in combination for 24 hours. Total cellular RNA was extracted, and mRNAs for Npnt and Gapdh were examined using real-time PCR analysis. (C) MC3T3-E1 cells were pretreated with or without Pkcα siRNA (20 nM) for 24 hours, and then treated with PMA (10 nM) alone or in combination for 24 hours. Proteins were extracted and subjected to western blotting to detect Pkcα and actin. (D) Total cellular RNA was extracted, and mRNAs for Npnt and Gapdh were examined using real-time PCR analysis. Results are shown as the mean ± SD of three samples. **P <0.01, Student's t-test, as compared to presence or absence of PMA, Gö6983, and Pkcα siRNA.