FGF‐2 suppresses expression of nephronectin via JNK and PI3K pathways

Nephronectin (Npnt), an extracellular matrix protein, is a ligand for integrin α8β1 and is involved in the development of various organs, such as the kidneys, bones, liver, and muscles. Previously, we found that Npnt expression was inhibited by various cytokines including transforming growth factor‐β (Tgf‐β) and oncostatin M (Osm). Fibroblast growth factor (Fgf)‐2, otherwise known as basic Fgf, also plays important roles in skeletal development and postnatal osteogenesis. In this study, Npnt expression was found to be suppressed by Fgf‐2 in MC3T3‐E1 cells, an osteoblast‐like cell line, in a dose‐ and time‐dependent manners. Furthermore, Fgf‐2‐mediated Npnt mRNA suppression was shown to involve the Jun N‐terminal kinase (JNK) and phosphoinositide‐3 kinase (PI3K) pathways. Together, our results suggest that FGF‐2 suppresses Npnt gene expression via JNK and PI3K pathways.

Nephronectin (Npnt), an extracellular matrix protein, is a ligand for integrin a8b1 and is involved in the development of various organs, such as the kidneys, bones, liver, and muscles. Previously, we found that Npnt expression was inhibited by various cytokines including transforming growth factor-b (Tgf-b) and oncostatin M (Osm). Fibroblast growth factor (Fgf)-2, otherwise known as basic Fgf, also plays important roles in skeletal development and postnatal osteogenesis. In this study, Npnt expression was found to be suppressed by Fgf-2 in MC3T3-E1 cells, an osteoblast-like cell line, in a dose-and time-dependent manners. Furthermore, Fgf-2mediated Npnt mRNA suppression was shown to involve the Jun N-terminal kinase (JNK) and phosphoinositide-3 kinase (PI3K) pathways. Together, our results suggest that FGF-2 suppresses Npnt gene expression via JNK and PI3K pathways.
Nephronectin (Npnt), identified in osteoblast-like MC3T3-E1 cells, is an extracellular protein with an epidermal growth factor-like repetitive structure. Moreover, Npnt is the primary ligand for a8b1 integrin and plays an important role in regulation of cell adhesion, differentiation, and spreading, as well as survival of various organs, such as the kidneys, bones, liver, and muscles [1]. In vivo, Npnt expression is particularly prominent at epithelial-mesenchymal interfaces in tissues undergoing morphogenesis [2,3]. Therefore, elucidation of how the Npnt gene is expressed is important for understanding the association between bone development and cell adhesion [4,5].
MC3T3-E1 is a cloned mouse osteoblast-like cell line that retains the synthetic functions of bone and has been utilized as an in vitro bone model of development systems. Using MC3T3-E1 cells, Kahai et al. [4] showed that some endogenous miRNAs might repress Npnt expression, resulting in a lower level of osteoblast differentiation. Other studies have also reported that transforming growth factor-b (Tgf-b) and oncostatin M (Osm) downregulate Npnt expression in both dose-and time-dependent manners, while osteoblast differentiation induced by Npnt was found to be inhibited by Tgf-b and Osm in MC3T3-E1 cells [6,7].
Results of phylogenetic analysis suggested that 22 different Fgf genes can be arranged into seven subfamilies containing 2-4 members each [8]. In another study, various growth factors, including fibroblast growth factor-2 (Fgf-2), transforming growth factor-(Tgf-b), insulin-like growth factor-1 (Igf-1), platelet-derived growth factor, and prostaglandin E 2 , were shown to act as autocrine and paracrine hormones for regulation of bone cell proliferation [9]. Fgf-2, which is stored in the extracellular matrix and expressed in osteoblasts [10], influences proliferation and differentiation of a variety of cell types in vitro [11,12]. In bone cell culture experiments, Fgf-2 showed increased replication and reduced differentiation markers, such as alkaline phosphatase and type I collagen [13][14][15], thus is considered to have important functions in bone homeostasis.
This study was designed to examine the effects of Fgf-2 on expression of Npnt and related molecular mechanisms. Our results revealed that Npnt expression in MC3T3-E1 cells is regulated by Fgf-2 via the Jun N-terminal kinase (JNK) and phosphoinositide-3 kinase (PI3K) pathways.
Real-time PCR was performed using a StepOne TM Realtime PCR System (Applied Biosystems, Waltham, MA, USA) with SYBR Green Fast PCR Master Mix (Applied Biosystems) with the following specific PCR primers: Gapdh, 5 0 -AAATGGTGAAGGTCGGTGTG-3 0 and 5 0 -TG AAGGGGTCGTTGATGG-3 0 ; and Npnt, 5 0 -CACGAGTA ATTACGGTTGACAACAG-3 0 and 5 0 -CTGCCGTGGAA TGAACACAT-3 0 . The total reaction volume was 10 lL including 2 lL of a cDNA sample. The thermos-cycling parameters employed were holding for 20 s at 95°C, followed by 40 cycles of denaturation at 95°C for 1 min, and annealing and extension at 60°C for 20 s. Amplified products were determined using a standard curve analysis, and the expression level of each gene was normalized against that of Gapdh and expressed as the relative value for each experiment.

Western blotting
Cell lysates were collected using sample buffer solution with reducing reagent (69)

Statistical analysis
All results are expressed as the mean AE standard deviation (SD). For results shown in Figs 1A, 2A, 3B and 4A,B, statistical analysis was performed using one-way ANOVA, while those shown in Figs 2B and S1 were analyzed using a two-tailed Student's t-test. A P value of < 0.05 or < 0.01 was considered to indicate statistical significance.

Fgf-2 strongly suppressed Npnt expression in Fgfs
We attempted to determine whether the expression of Npnt is regulated by members of the Fgf family, including Fgf-1, Fgf-2, Fgf-8b, Fgf-9, and Fgf-23. MC3T3-E1 cells were exposed to each of the investigated Fgfs for 24 h, and then, the level of Npnt mRNA was examined using real-time PCR analysis (Fig. 1A). Fgf-2 showed significant suppression of Npnt mRNA expression, while suppression by Fgf-1 was also noted, although the level was not as great as seen with Fgf-2. With 1 ngÁmL À1 of Fgf-2, the expression level of Npnt protein was also suppressed in MC3T3-E1 cells after 24 h of incubation (Fig. 1B). Thus, we exposed MC3T3-E1 cells to various doses of Fgf-2 for 24 h and found that downregulation of Npnt expression occurred in a dose-dependent manner, with the level reaching a plateau at~1 ngÁmL À1 (Fig. 2A). Finally, we investigated time-dependent suppression by Fgf-2 at a dose of 1 ngÁmL À1 and detected a significant decrease in Npnt mRNA at 6 h after its addition to culture (Fig. 2B). The rate of Npnt expression by cells treated with Fgf-2 as compared to untreated cells was decreased in a time-dependent manner, with the level reaching a plateau at 24 h (data not shown).

Expression of Fgfr genes in MC3T3-E1 cells and their involvement in Npnt expression
The Fgfr (Fgf receptor) gene family is comprised of four members, Fgfr1-Fgfr4 [8]. We found that each was well expressed in kidney specimens, while Fgfr1-3 were expressed in MC3T3-E1 cells (Fig. 3A). After treating MC3T3-E1 cells with BGJ398, an Fgfr inhibitor, real-time PCR analysis was performed, which showed that 0.1 lM of BGJ398 blocked suppression of Npnt mRNA induced by 1 ngÁmL À1 of Fgf-2 and restored its expression (Fig. 3B).

Discussion
This study is the first to show that Fgf-2 strongly inhibits Npnt mRNA expression in a manner related to the JNK and PI3K signaling pathways (Fig. 5). Npnt, which enhances osteoblast differentiation, is expressed in the basement membrane of developing teeth and extracellular matrix of developing jawbones [5]. In addition, Linton et al. [20] showed that embryos lacking a functional Npnt gene frequently display kidney agenesis or hypoplasia, which could be traced to a delay in invasion of metanephric mesenchyme by the ureteric bud at an early stage of kidney development. It has been speculated that kidney disease causes disordered mineral metabolism, resulting in bone disease and ultimately fracture [21]; thus, it is considered that Npnt is closely related to bone metabolism. The relationship of Fgf-2 with skeleton development and bone metabolism has  been reported in several studies. For example, overexpression of human FGF-2 in mice (TgFGF-2) results in dwarfism, with shortening and flattening of long bones and moderate macrocephaly [22], while its deletion has been shown to lead to decreased levels of bone mass, formation, and mineralization in mice [23,24].
A previous study also demonstrated that Fgfs activates PI3K signaling [8]; thus, we investigated whether Npnt mRNA expression suppressed by Fgf-2 is also regulated via the PI3K pathway. Several different materials, such as Igf-1 [25] and ghrelin [26], have been reported to activate PI3K signaling, while the present results also suggest that these substances may regulate Npnt mRNA expression.
We used the osteoblast-like MC3T3-E1 cell line in the present study and also examined primary osteoblasts obtained from calvaria of 1-day-old mice. Fgf-2 inhibited the expression of Npnt mRNA in primary osteoblasts (Fig. S1). It has also been reported that Npnt enhances osteoblast differentiation, while contrasting findings showed that FGF-2 increases osteoblast differentiation and extracellular matrix mineralization in vitro [27-29]. Similar contradictory results were shown in another study, which found that expression of a transcriptional co-activator with a PDZ-binding motif (Taz) was regulated by Fgf-2 [30]. These results might help to explain the complex mechanisms of Fgfs.

Conclusions
Fibroblast growth factor-2 suppresses Npnt mRNA expression in MC3T3-E1 cells in a dose-and timedependent manner by activation of the JNK and PI3K

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Fig. S1. Fgf-2 inhibits expression of Npnt in primary osteoblasts. Real-time PCR analysis was performed using cDNA from primary osteoblasts after treatment with 1 ng.mL À1 of Fgf-2 for 24 hours. Values are shown as the mean AE SD of 3 samples as compared to the value without Fgf treatment. *p<0.05, **p<0.01; relative to level in cells without treatment (Student's t-test).