Cryptochrome 1 Regulates Osteoblast Differentiation via the AKT Kinase and Extracellular Signal-Regulated Kinase Signaling Pathways

Abstract

The many circadian clock genes build up a network structure that controls physiological processes, such as the sleep cycle, metabolism, and hormone secretion. Cryptochrome 1 (CRY1), as one of the critical circadian proteins, is closely related to bone formation. However, the regulatory function of CRY1 in osteogenic differentiation remains unclear. In this study, we investigated the role of CRY1 in regulating proliferation and osteoblast differentiation in C3H10 and C2C12 cells after silencing Cry1 using short hairpin RNA interference. In vitro experiments confirmed that the expression level of CRY1 gradually increased during the osteogenic differentiation process, and Cry1 knockdown inhibited the proliferation and differentiation of osteoblastic cells. In addition, Cry1 knockdown inhibited the phosphorylation of AKT kinase (AKT) and extracellular signal-regulated kinase (ERK), which suppressed the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-AKT and mitogen-activated protein kinase (MAPK)-ERK signaling pathways. Taken together, these findings show that CRY1 regulates the proliferation and differentiation of osteoblastic cells in an AKT and ERK-dependent manner.

Introduction

Bone constantly undergoes remodeling to adapt to environmental mechanical stress, for stable calcium and phosphorous metabolism, and to repair bone damage (Fujisawa et al., 2018). Bone remodeling depends on the balance between bone formation and resorption (Eleniste et al., 2016). Osteoblasts differentiate into osteocytes, and during the processes of proliferation, differentiation, and maturation, they excrete a number of osteogenic markers and minerals, which have been identified as requisite factors to regulate bone formation and to regulate and activate osteoclasts for bone absorption (Xiong et al., 2018). These bone-specific markers include alkaline phosphatase (ALP), runt-related transcript factor 2 (RUNX2), osterix (OSX), collagen type I α1 (COL1A1), osteocalcin (OCN), osteoprotegerin (OPG), and osteopontin (OPN) (Sprague et al., 2016; Tise et al., 2016; Yang et al., 2016).

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-AKT kinase (AKT) signaling pathway and mitogen-activated protein kinase (MAPK)–extracellular signal-regulated kinase (ERK) signaling pathways have been identified as important regulatory pathways during osteogenic differentiation (Srivastava et al., 2014; Wu et al., 2014; Xi et al., 2014). The PI3K-AKT pathway is activated by growth factors and other extracellular signals to regulate cell growth, proliferation, and survival. Some researchers reported that this pathway is activated by RUNX2 and OSX during bone formation (Choi et al., 2011, 2014). Other studies demonstrated that the MAPK/ERK pathway regulates both ex vivo osteogenesis and in vivo bone formation (Wang et al., 2011; Wu et al., 2014).

In addition, bone morphogenetic protein 2 (BMP-2), a strong regulator of bone formation, exerts its osteogenic capacity via phosphorylated AKT (p-AKT) and ERK (p-ERK) (Mukherjee and Rotwein, 2009; Wu et al., 2014).

Cryptochrome 1 (CRY1), one of the critical circadian clock proteins, plays an important role in circadian clock and clock-related diseases (Gauger and Sancar, 2005; Kelleher et al., 2014). The circadian clock exists in almost all eukaryotic organisms, and controls the expression of ∼10% of genes, and regulates cell metabolism, proliferation, DNA damage repair, apoptosis, and autophagy (Delaunay et al., 2002; Shearman et al., 2000). Numerous studies have demonstrated the important role of circadian genes in bone remodeling (Lieben, 2016; Xu et al., 2016). Mice lacking Cry genes displayed a high bone mass (Fu et al., 2005); blue laser-irradiated mesenchymal stem cells (MSCs) showed enhanced extracellular calcification and decreased messenger RNA (mRNA) levels of Cry1 (Kushibiki and Awazu, 2009).

These findings indicated that CRY1 is closely related to the functions of osteoblasts. However, its exact role and how CRY1 regulates osteogenesis remains unclear.

Previously, we found that CRY1 promotes osteogenic differentiation of human osteoblastic cells via the Wnt/β-Catenin signaling pathway (Zhou et al., 2018). In the present research, we detected the function of CRY1 in the mouse MSCs C3H10 and in mouse multipotent mesenchymal progenitor cells C2C12. We found that Cry1 knockdown inhibited the expression of bone-specific markers and attenuated osteoblast differentiation, which was consistent with our preliminary research results. Furthermore, the function of CRY1 in mouse osteoblasts was mediated through the PI3K-AKT and MAPK-ERK signaling pathways. Our findings increase our understanding of the roles of CRY1 in osteogenic differentiation.

Materials and Methods

Reagents and antibodies

Ascorbic acid (AA), β-glycerophosphate (β-GP), dexamethasone (DXMS), and Alizarin Red S (AR-S) were purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies included those recognizing CRY1 (ab104736; Abcam, Cambridge, MA), AKT1 (sc-1618; Santa Cruz Biotechnology, Santa Cruz, CA), p-AKT (4060; Cell Signaling Technology, Danvers, MA), mouse double minute 2 (MDM2; sc-965; Santa Cruz Biotechnology), p-MDM2 (35215; Cell Signaling Technology), P53 (sc-126; Santa Cruz Biotechnology), P21 (2947; CST), cyclin-dependent kinase 2 (CDK2; sc-163; Santa Cruz Biotechnology), Cyclin E (sc-247; Santa Cruz Biotechnology), Cyclin A (sc-751; Santa Cruz Biotechnology), and β-actin (a1978; Sigma-Aldrich). Secondary antibodies include anti-mouse IgG and anti-rabbit IgG (4408, 4414; Cell Signaling Technology).

Construction and identification of lentivirus short hairpin RNA plasmids

A three-plasmid system comprising pSPAX2, pMD2G, and pHB-U6-MCS-CMV-ZsGreen-PGK-PURO was used for lentivirus packing to express short hairpin RNAs (shRNAs), which was obtained from Hanbio Biotechnology Co. (Shanghai, China). To knock down Cry1, three Cry1 RNA interference (RNAi) sequences were selected based on the GenBank mRNA encoding mouse CRY1 (NM_007771; Cry1-shRNA1, CAAGTGTTTGATAGGAGTT; Cry1-shRNA2, GCCACCTCTAACATATAAA; Cry1-shRNA3, ATCAGTGTTTGATCTAATT). Then, three shRNA targeting mouse CRY1 were designed (Table 1) and inserted into the lentiviral vector pHB-U6-MCS-CMV-ZsGreen-PGK-PURO.

Table 1.

Sequences of CRY1-shRNAs Used in the Present Study

Group	Sense strand	Antisense strand
CRY1-shRNA1	5′-CAAGTGTTTGATAGGAGTT-3′	5′-AACTCCTATCAAACACTTGGC-3′
CRY1-shRNA2	5′-GCCACCTCTAACATATAAA-3′	5′-TTTATATGTTAGAGGTGGCTG-3′
CRY1-shRNA3	5′-ATCAGTGTTTGATCTAATT-3′	5′-AATTAGATCAAACACTGATGT-3′
Control-shRNA	5′-TTCTCCGAACGTGTCACGT-3′

CRY1, cryptochrome 1; shRNA, short hairpin RNA.

Lentivirus production and transduction

To produce lentiviruses, lentiviral vectors pMD2G, psPAX2, and pHB-U6-MCS-CMV-ZsGreen-PGK-PURO were transfected into 293T cells (at 50%–70% cell density) using LipoFiter^™ (Hanbio Biotechnology Co.) following the manufacturer's instructions. After incubation for 12 hours, the culture medium was replaced with complete high-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S; Invitrogen, Carlsbad, CA). At 48 and 72 hours later, cell supernatants were gathered and filtered using 0.45-μm cellulose acetate filters (Millipore, Billerica, MA). Four kinds of lentivirus were stored at −80°C.

C3H10 and C2C12 cells were seeded into six-well plates and lentivirus was added at 3 or 4 μg/mL in polybrene (Beyotime, Jiangsu, China) for 12 hours and then changed with ordinary culture medium for 2 days. To get stable CRY1 knockdown cells, cells were cultured with DMEM in the presence of 4 μg/mL of puromycin (Sigma-Aldrich) for 3 days, and then changed to 1 μg/mL of puromycin for the next 7 days. The cells were separated into three groups, respectively: the experimental group expressing CRY1-shRNA1–3, the control shRNA group, and the blank cell group.

Cell culture and osteogenic induction

C3H10, C2C12, and 293T cells were purchased from ATCC (Manassas, VA) and cultured in DMEM containing 1% P/S and 10% FBS. The culture medium for osteogenic differentiation was ordinary DMEM plus 10 mM β-GP, 50 μg/mL AA, and 0.1 μM DXMS; the differentiation medium was replaced every 2 days.

Cell counting kit 8 assay

The effect of CRY1 on the viability of C3H10 and C2C12 cells was evaluated using a Cell Counting Kit 8 (CCK-8) (Dojindo, Kumamoto, Japan) in accordance with manufacturer's instructions. Cells were digested and seeded at 1 × 10³ cells/well into 96-well plates with five replicates. The cells were cultured for 24, 48, 72, and 96 hours, then 10 μL of CCK-8 reagent was added per well and incubated at 37°C for 2 hours. The absorbance was detected at 450 nm using a Tecan Infinite 200 PRO multi-well plate reader (Tecan Ltd., Männedorf, Switzerland) and expressed in optical density units. The experiment was performed in triplicate.

ALP staining and AR-S staining

Ay 0, 3, 7, and 14 days after induction with osteogenic differentiation medium, ALP staining was monitored using an ALP staining kit (DE0004; Leagene, Beijing, China) according to the manufacturer's protocol. For AR-S staining (Sigma-Aldrich), cells were stained with 50 mM AR-S for 1 hour at 37°C after fixed by 4% paraformaldehyde for 3 minutes at 37°C. After staining, the cells were rinsed with phosphate-buffered saline (PBS), air dried, and photographed.

ALP activity assay

An ALP activity kit (Beyotime) was used to detect ALP activity in accordance with manufacturer's instructions. The concentration of total protein was assayed using a bicinchoninic acid (BCA) method (Beyotime) and used to normalize the relative ALP activity.

Western blotting analysis

Logarithmic phase C3H10 and C2C12 cells were washed twice with PBS and added with Radioimmunoprecipitation assay (RIPA) buffer and then collected using a cell scraper. Cell lysates were vibrated 10 seconds every 5 minutes for 30 minutes at 0°C and subjected to centrifugation for 10 minutes (12,000 rpm, 4°C), and then the supernatants were collected into clean EP tubes. The concentration of total protein was quantified using the BCA method in accordance with the protocol (Beyotime). The extracted protein solution (10 μg protein) was mixed with 5 × loading buffer and heated at 100°C for 10 minutes. The samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for about 90 minutes, and then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore).

The PVDF membranes were blocked in 5% nonfat milk for 2 hours at 20°C. The PVDF membranes were then probed with primary antibodies at 4°C overnight. The PVDF membranes were then washed three times for 5 minutes each in Tris-buffered saline Tween-20, and then incubated with secondary antibodies at 37°C for 2 hours. An ECL Detection Kit (Share-Bio, Zhejiang, China) was used to detect the immunoreactive proteins and the membranes were photographed. The images were analyzed the FluorChem E system (ProteinSimple, San Jose, CAA). The assays were performed three times.

Quantitative real-time polymerase chain reaction

We used RNAiso Plus (Takara, Shiga, Japan) for the overall RNA isolation from C3H10 and C2C12 cells. The RNA concentration and quality were determined using the Tecan Infinity 200 PRO multiwell plate reader (Tecan Ltd.) to detect absorbance at 260 and 280 nm. The total RNAs were reverse transcribed by PrimeScript^™ RT Master Mix Kit at 37°C for 15 minutes and 85°C for 5 seconds, according to the manufacturer's instructions. The quantitative real-time polymerase chain reaction (qRT-PCR) primers for Cry1, Alp, Runx2, Osx, Col1a1, Ocn, Opg, Opn, and Actb (beta-actin) were obtained from PrimerBank and are listed in Table 2.

Table 2.

Primer Sequences Used in the Present Study

Gene	Forward	Reverse
CRY1	5′-CACTGGTTCCGAAAGGGACTC-3′	5′-CTGAAGCAAAAATCGCCACCT-3′
ALP	5′-ACTGGGGCCTGAGATACCC-3′	5′-CCCGCTTTAACCAGTGCAAC-3′
RUNX2	5′-AGTCCGTCTTCTGCCGTATCA-3′	5′-AGCTTCGTAACCCGAATGGAT-3′
OSX	5′-GGAAAGGAGGCACAAAGAAGC-3′	5′-CCCCTTAGGCACTAGGAGC-3′
COL1A1	5′-GTGCGATGACGTGATCTGTGA-3′	5′-CGGTGGTTTCTTGGTCGGT-3′
OCN	5′-CACTCCTCGCCCTATTGGC-3′	5′-CCCTCCTGCTTGGACACAAAG-3′
OPG	5′-GAAGTTTCGCAGACCTGACAT-3′	5′-GTATGCACCATTCAACTCCTCG-3′
OPN	5′-TGCTTGTGACGAGCTATCAG-3′	5′-GAGGACAGGGAGGATCAAGT-3′
β-actin	5′-GGGACCTGACTGACTACCTC-3′	5′-TCATACTCCTGCTTGCTGAT-3′

ALP, alkaline phosphatase; COL1A1, collagen type I α1; OCN, osteocalcin; OPG, osteoprotegerin; OPN, osteopontin; OSX, osterix; RUNX2, runt-related transcript factor 2.

SYBR Premix Ex Taq^™ (Takara) was used for qRT-PCR. An Applied Biosystems 7500 fast RT-PCR instrument (Thermo Fisher Scientific, Waltham, MA) was used to amplify the complementary DNA (cDNA) samples. The reaction conditions comprised predenaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and t 55°C for 34 seconds. The relative expression levels of osteoblast-specific marker mRNAs were normalized based on the expression of Actb. The data were calculated and analyzed based on the 2^−ΔΔCT method. The qRT-RCR assays were performed for three times.

Statistical analysis

All data were analyzed using the Statistical Package for Social Sciences (SPSS) 21.0 (IBM Corp., Armonk, NY). Data are represented as the mean ± standard deviation. A least significant difference test was used for the comparison of data between two groups. Comparisons of data among groups were calculated using one-way analysis of variance. p < 0.05 was considered statistically significant.

Results

Osteogenic differentiation induces CRY1 expression

First, we examined the endogenous CRY1 protein levels in C3H10 and C2C12 cells using western blotting. Both C3H10 and C2C12 cells highly expressed the CRY1 protein (Fig. 1A). Then, to evaluate the relationship between CRY1 and osteoblast differentiation, the expression mode of CRY1 in C3H10 cells cultured with osteogenic differentiation medium (containing AA, β-GP, and DXMS) was detected. The results showed that with increasing incubation time, the protein and mRNA levels of CRY1 gradually increased, together with enhanced osteogenesis and bone mineralization (Figs. 1B and 2B).

FIG. 1.

Expression of CRY1 during osteogenic differentiation. (A) Endogenous expression of CRY1 in C3H10 and C2C12 cells, as assessed using western blotting. (B) The expression pattern of CRY1 in C3H10 cells during osteogenic differentiation, as assessed using western blotting. (C) ALP staining and relative ALP activity assay of C3H10 cells. (D) AR-S staining for bone nodules in C3H10 cells; the AR-S staining activity was quantified by densitometry at 562 nm. Values are expressed as the mean ± SD. **p < 0.01 versus the control. ***p < 0.005 versus the control. ALP, alkaline phosphatase; AR-S, Alizarin Red S; CRY1, cryptochrome 1; SD, standard deviation.

FIG. 2.

Association of CRY1 with osteogenesis. (A) CRY1 protein and mRNA levels in C2C12 cells induced to undergo osteogenic differentiation. (B) Cry1 mRNA levels of C3H10 and C2C12 cells during osteoblast differentiation. (C–I) Expression pattern of osteogenic markers after osteogenic induction. (J) ALP staining and relative ALP activity assay of C2C12 cells. (K) AR-S staining for bone nodules in C2C12 cells; the AR-S staining activity was quantified by densitometry at 562 nm. Values are expressed as the mean ± SD. *p < 0.05 versus the control. **p < 0.01 versus the control. ***p < 0.005 versus the control. mRNA, messenger RNA.

For further verification, we repeated the above experiments in C2C12 cells. As shown in Figure 2A and B, the expression levels of CRY1 were enhanced during osteoblast differentiation. In addition, ALP and AR-S staining and qRT-PCR for detecting the mRNA expression levels of osteogenesis-specific markers Alp, Runx2, Osx, Col1a1, Ocn, Opg, and Opn were performed. Both the ALP and AR-S staining and expression of osteogenic markers were enhanced as differentiation increased (Fig. 2C–K). Taken together, these results indicated that CRY1 plays an important role in osteogenic differentiation and mineralization.

Expression levels of CRY1 protein and mRNA in C3H10 and C2C12 cells

On the basis of the high primary CRY1 expression level displayed in Figure 1A, we established Cry1-silenced cell lines (C3H10-CRY1-shRNA3 and C2C12-CRY1-shRNA2), as well as the corresponding control-shRNA cell lines (Fig. 3C).

FIG. 3.

Establishment of Cry1 knockdown in C3H10 and C2C12 cells. (A, B) Western blotting results of blank groups, control-shRNA groups, and CRY1-shRNA groups in C3H10 and C2C12 cells. (C—F) Measurement of the CRY1 silencing efficiency at the protein and mRNA levels in C3H10 and C2C12 cells. Values are expressed as the mean ± SD. *p < 0.05 versus the control. **p < 0.01 versus the control. ***p < 0.005 versus the control. shRNA, short hairpin RNA.

Data from western blotting and qRT-PCR assays displayed the relative protein and RNA levels of CRY1, as normalized to the level of β-actin in the blank C3H10 and C2C12 cells, control-shRNA group, and CRY1-shRNA1–3 groups (Fig. 3C–F). Both the mRNA and protein levels were most significantly decreased in the CRY1-shRNA3 group in C3H10 cells and by CRY1-shRNA2 in the C2C12 cells (p < 0.05); therefore, these shRNAs were chosen for further use.

Silencing of Cry1 inhibits the proliferation and osteogenic differentiation of C3H10 and C2C12 cells

To investigate the potential role of CRY1 in the process of osteogenesis, we detected the proliferation rate, expression levels of osteogenesis markers, and ALP and AR-S staining. The CCK-8 assay demonstrated that the proliferation rates of the CRY1-shRNA groups were lower than the control-shRNA groups in both C3H10 and C2C12 cells (Fig. 4A, B, p < 0.05). We then examined osteogenic differentiation markers in the two cell lines using qRT-PCR. The results demonstrated Cry1 knockdown significantly downregulated the expression levels of these markers (Fig. 4C–P, p < 0.05). ALP and AR-S staining were performed to evaluate the function of CRY1 in osteogenesis and mineralization. Figure 4Q and R show that differentiation and mineralization were strongly inhibited in CRY1-shRNA group cells, compared with that in the control group (p < 0.05).

FIG. 4.

Cry1 knockdown inhibits the proliferation and differentiation abilities of osteoblasts. (A, B) Proliferation of C3H10 and C2C12 cells. Relative absorbance of cells in a CCK-8 assay was determined at 12-hour intervals. (C–P) Cry1 silencing inhibited the expression of osteogenic specific markers in C3H10 and C2C12 cells. (Q, R) Cry1 silencing inhibited ALP activity and mineralized matrix formation of C3H10 and C2C12 cells, as determined using ALP staining, relative ALP activity, and AR-S staining. Values are expressed as the mean ± SD. *p < 0.05 versus the control. **p < 0.01 versus the control. ***p < 0.005 versus the control. CCK-8, Cell Counting Kit 8.

Cry1 knockdown decreases AKT and ERK activation

To identify the exact mechanism of Cry1 silencing in regulating proliferation and osteogenic differentiation of C3H10 and C2C12 cells, we detected the changes in the expression levels of AKT and ERK signaling-related molecules. As shown in Figure 5A, the levels of p-AKT (S473), p-AKT (T308), and p-ERK were significantly enhanced in the control group, in accordance with the high expression levels of CRY1 (p < 0.05). Then, we detected these signals after osteogenic differentiation for 0, 3, 7, and 14 days and found that they were increasingly activated in the control groups, but continuously inhibited in the CRY1-shRNA groups (Fig. 5B, C, p < 0.05). Therefore, we hypothesized that AKT signaling and ERK signaling were the main downstream signaling pathways of CRY1-regulated osteogenesis.

FIG. 5.

Cry1 knockdown decreases AKT and ERK activation. (A) AKT and ERK activation was inhibited after Cry1 silencing in C3H10 and C2C12 cells. (B, C) Representative immunoblots for AKT and ERK levels in control groups and CRY1-shRNA groups during osteogenic induction. (D–K) Measurement of the gray intensity of the immunoreactive protein bands in (A–C). Values are expressed as the mean ± SD. **p < 0.01 versus the control. ***p < 0.005 versus the control. AKT, AKT kinase; ERK, extracellular signal-regulated kinase.

Discussion

Given that Cry^−/− mice showed high bone mass, extracellular calcification, and the mRNA levels of Cry1 were deregulated in blue laser-irradiated MSCs (Fu et al., 2005; Kushibiki and Awazu, 2009), we speculated that CRY1 regulates osteoblast differentiation.

Therefore, our observation that CRY1 is functionally expressed in osteoblast cells, which have the osteogenic differentiation ability, is important. As one of the key circadian clock genes, CRY1 functions on the basis of the canonical feedback loop of seven core circadian proteins: clock circadian regulator (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), which act as transcriptional activators of cryptochromes (CRY1, CRY2) and the period proteins (PER1, PER2, PER3). Once the CRY/PER complex is formed, it represses the transcription of CLOCK and BMAL1 (Honma et al., 2002; Preitner et al., 2002). In mammals, ∼10% of all genes are clock-controlled genes, including certain osteogenesis-related genes (Delaunay and Laudet, 2002; Shearman et al., 2000).

We found that CRY1 affects proliferation and osteogenic differentiation (Zhou et al., 2018); therefore, we analyzed common osteogenesis-related signaling pathways and found that CRY1 may function through the AKT and ERK signaling pathways. AKT, also named protein kinase B, is a Ser/Thr kinase that affects cell proliferation by inducing cell survival and metastasis signals, and influences osteogenic differentiation by enhancing the expression of BMP-2, RUNX2, and OSX when phosphorylated (Choi et al., 2014; Mukherjee and Rotwein, 2009; Risso et al., 2015). ERK mediates BMP-2 and RUNX2-induced osteoblast differentiation, and modulates the fate of osteoblastic cells when exposed to mitogenic stimulation or serum starvation stress, together with AKT (Almeida et al., 2005).

These studies demonstrated the important role of AKT and ERK signaling in osteoblast differentiation. Our data revealed that AKT and ERK were gradually activated in control groups, but significantly inhibited in CRY1-shRNA groups. These results not only supported the hypothesis that AKT and ERK participate in the proliferation and differentiation of osteoblasts but also provided evidence that CRY1 regulates osteogenic differentiation via the AKT and ERK signaling pathways.

In conclusion, the present study revealed that CRY1, as one of the key circadian genes and a novel regulator of proliferation and differentiation of osteoblasts, controls osteogenic differentiation via the AKT and ERK signaling pathways. These findings may lead to the development of therapeutic strategies through gene modification to regulate bone formation. However, further research is needed to reveal the exact mechanisms of CRY1's regulation of osteogenesis.

Footnotes

Acknowledgments

This work was supported by the Key Department of Minhang District (2017MWTZ02); the Key Department of the Fifth People's Hospital of Shanghai (2017WYZDZK02); the Natural Science Foundation of Minhang District of Shanghai (2017MHZ15); the Fifth People's Hospital of Shanghai, Fudan University (2018WYZT01); and the Minhang District Leading Talent Development Funds.

Author Disclosure Statement

The authors declare there are no financial conflicts of interest.

References

Almeida

, Han

, Bellido

, Manolagas

S.C.

, and Kousteni

(2005). Wnt proteins prevent apoptosis of both uncommitted osteoblast progenitors and differentiated osteoblasts by beta-catenin-dependent and —independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase/AKT. J. Biol. Chem. 280, 41342–41351.

Choi

Y.H.

, Jeong

H.M.

, Jin

Y.H.

, Li

, Yeo

C.Y.

, and Lee

K.Y.

(2011). Akt phosphorylates and regulates the osteogenic activity of Osterix. Biochem. Biophys. Res. Commun. 411, 637–641.

Choi

Y.H.

, Kim

Y.J.

, Jeong

H.M.

, Jin

Y.H.

, Yeo

C.Y.

, and Lee

K.Y.

(2014). Akt enhances Runx2 protein stability by regulating Smurf2 function during osteoblast differentiation. FEBS J. 281, 3656–3666.

Delaunay

, and Laudet

(2002). Circadian clock and microarrays: Mammalian genome gets rhythm. Trends Genet. 18, 595–597.

Eleniste

P.P.

, Patel

, Posritong

, Zero

, Largura

, Cheng

Y.H.

, Himes

E.R.

, Hamilton

, Baughman

, Kacena

M.A.

, and Bruzzaniti

(2016). Pyk2 and megakaryocytes regulate osteoblast differentiation and migration via distinct and overlapping mechanisms. J. Cell. Biochem. 117, 1396–1406.

, Patel

M.S.

, Bradley

, Wagner

E.F.

, and Karsenty

(2005). The molecular clock mediates leptin-regulated bone formation. Cell, 122, 803–815.

Fujisawa

, Hara

, Takami

, Okada

, Matsumoto

, Yamamoto

, and Sakaida

(2018). Evaluation of the effects of ascorbic acid on metabolism of human mesenchymal stem cells. Stem Cell Res. Ther. 9, 93.

Gauger

M.A.

, and Sancar

(2005). Cryptochrome, circadian cycle, cell cycle checkpoints, and cancer. Cancer Res. 65, 6828–6834.

Honma

, Kawamoto

, Takagi

, Fujimoto

, Sato

, Noshiro

, Kato

, and Honma

(2002). Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature, 419, 841–844.

10.

Kelleher

F.C.

, Rao

, and Maguire

(2014). Circadian molecular clocks and cancer. Cancer Lett. 342, 9–18.

11.

Kushibiki

, and Awazu

(2009). Blue laser irradiation enhances extracellular calcification of primary mesenchymal stem cells. Photomed. Laser Surg. 27, 493–498.

12.

Lieben

(2016). Bone: The circadian clock controls bone remodelling. Nat. Rev. Rheumatol. 12, 132.

13.

Mukherjee

, and Rotwein

(2009). Akt promotes BMP2-mediated osteoblast differentiation and bone development. J. Cell Sci. 122, 716–726.

14.

Preitner

, Damiola

, Lopez-Molina

, Zakany

, Duboule

, Albrecht

, and Schibler

(2002). The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell, 110, 251–260.

15.

Risso

, Blaustein

, Pozzi

, Mammi

, and Srebrow

(2015). Akt/PKB: One kinase, many modifications. Biochem. J. 468, 203–214.

16.

Shearman

L.P.

, Sriram

, Weaver

D.R.

, Maywood

E.S.

, Chaves

, Zheng

, Kume

, Lee

C.C.

, van der Horst

G.T.

, Hastings

M.H.

, and Reppert

S.M.

(2000). Interacting molecular loops in the mammalian circadian clock. Science, 288, 1013–1019.

17.

Sprague

S.M.

, Bellorin-Font

, Jorgetti

, Carvalho

A.B.

, Malluche

H.H.

, Ferreira

, D'Haese

P.C.

, Drueke

T.B.

, Du

, Manley

, Rojas

, and Moe

S.M.

(2016). Diagnostic accuracy of bone turnover markers and bone histology in patients with CKD treated by dialysis. Am. J. Kidney Dis. 67, 559–566.

18.

Srivastava

, Sharma

, Kumar

, and Roy

(2014). Bradykinin regulates osteoblast differentiation by Akt/ERK/NfkappaB signaling axis. J. Cell. Physiol. 229, 2088–2105.

19.

Tise

, Ferrante

, Mora

, and Tagliabracci

(2016). A biochemical approach for assessing cutoffs at the age thresholds of 14 and 18 years: A pilot study on the applicability of bone specific alkaline phosphatase on an Italian sample. Int. J. Legal Med. 130, 1149–1158.

20.

Wang

, Harimoto

, Liu

, Guo

, Hinshaw

, Chang

, and Wang

(2011). Spata4 promotes osteoblast differentiation through Erk-activated Runx2 pathway. J. Bone Miner. Res. 26, 1964–1973.

21.

, Chim

S.M.

, Kuek

, Lim

B.S.

, Chow

S.T.

, Zhao

, Yang

, Rosen

, Tickner

, and Xu

(2014). HtrA1 is upregulated during RANKL-induced osteoclastogenesis, and negatively regulates osteoblast differentiation and BMP2-induced Smad1/5/8, ERK and p38 phosphorylation. FEBS Lett. 588, 143–150.

22.

, Wai

, DeMambro

, Rosen

C.J.

, and Clemmons

D.R.

(2014). IGFBP-2 directly stimulates osteoblast differentiation. J. Bone Miner. Res. 29, 2427–2438.

23.

Xiong

, Almeida

, and O'Brien

C.A.

(2018). The YAP/TAZ transcriptional co-activators have opposing effects at different stages of osteoblast differentiation. Bone, 112, 1–9.

24.

, Ochi

, Fukuda

, Sato

, Sunamura

, Takarada

, Hinoi

, Okawa

, and Takeda

(2016). Circadian clock regulates bone resorption in mice. J. Bone Miner. Res. 31, 1344–1355.

25.

Yang

, Wu

, Chen

, Zhang

, You

, Yin

, Li

C.H.

, and Guan

Y.Q.

(2016). Effects of differentiation and antisenescence from BMSCs to hepatocy-like cells of the PAAm-IGF-1/TNF-alpha biomaterial. ACS Appl. Mater. Interfaces, 8, 26638–26647.

26.

Zhou

, Zhang

, Sun

, Yu

, and Wang

(2018). Cryptochrome 1 promotes osteogenic differentiation of human osteoblastic cells via Wnt/beta-Catenin signaling. Life Sci. 212, 129–137.