Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways

  • Authors:
    • Zi-Li Yu
    • Dian-Qi Li
    • Xiang-Yu Huang
    • Xin Xing
    • Ru-Qing Yu
    • Zhi Li
    • Zu-Bing Li
  • View Affiliations

  • Published online on: January 7, 2016     https://doi.org/10.3892/ijmm.2016.2450
  • Pages: 468-474
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Lysophosphatidic acid (LPA) is an efficient, bioactive phospholipid involved in various biological processes. In this study, LPA-induced connective tissue growth factor (CTGF/CCN2) expression and the underlying mechanisms were investigated using the MC3T3-E1 cell line. The MC3T3-E1 cells were stimulated with an inhibitor of LPA receptors, an activator and inhibitor of protein kinase C (PKC) and protein kinase A (PKA) for indicated periods of time. RT-qPCR and western blot analyses were used to measure the expression levels of CCN2. Immunofluorescence staining was used to observe the translocation of PKC. The mRNA expression level of CCN2 was increased following stimulation of the cells with LPA; LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation with LPA. This increase was accompanied by CCN2 protein synthesis. LPA receptor1/3 was inhibited by Ki16425, a specific inhibitor of LPA1/3; as a result, the LPA-induced increase in CCN2 expression was abrogated. LPA also induced the membrane translocation of PKC and enhanced PKC activity in the osteoblasts. Pre-treatment of the osteoblasts with staurosporine prevented the increase in CCN2 expression by induced by LPA, and the activation of PKC by phorbol 12-myristate 13-acetate (PMA) enhanced CCN2 expression, indicating that the PKC pathway is involved in the LPA-induced increase in CCN2 expression. The interference of PKA signaling also led to the induction of CCN2 expresion by LPA. These data indicate that LPA increases CCN2 expression through the activation of PKC and PKA. Thus, the regulatory functions of the PKC and PKA pathways are implicated in the LPA-induced increase in CCN2 expression.

Introduction

Lysophosphatidic acid (LPA) is an efficient, bioactive phospholipid involved in various biological processes, including cell proliferation, differentiation, apoptosis, adhesion, chemotaxis, survival and cancer cell invasion (16).

LPA is present in mammalian serum at concentrations of 1–5 µM; newly-formed LPA is rapidly released into the extracellular environment when platelets are activated (7). It has also been demonstrated that LPA is produced when P2X7 receptors expressed in osteoblasts are activated (8). Furthermore, LPA is released by osteoblasts (8), which suggests that LPA may be implicated in fracture healing.

LPA is a important signaling molecule exhibiting bioactive functions through receptors on the cytomembrane. At least 5 G-protein-coupled receptors (GPCRs) have been identified as LPA-specific receptors: LPA1, LPA2, LPA3, LPA4 and LPA5 (9,10). These receptors are expressed in different cell types, including osteoblasts, epithelial cells and skeletal muscle cells (2,1113).

Connective tissue growth factor (CTGF/CCN2) is a member of the CCN (Cyr61/CCN1, CTGF/CCN2 and NOV/CCN3) family, which also includes CCN4/WISP1, CCN5/WIPS2 and CCN6/WISP3 (14,15). CCN2 promotes the proliferation and differentiation of osteoblasts; CCN2 is also involved in bone development and fracture healing (1618).

LPA directly induces the production of CCN2 in epithelial cells, myoblasts and human renal fibroblasts (13,19,20) by binding to LPA receptors expressed in these cells. LPA receptors are also expressed in osteoblasts (2,3,11). However, whether LPA is capable of inducing changes in the levels of CCN2 in osteoblasts remains unclear. Thus, in the present study, we aimed to investigate the possible regulatory effects of LPA on CCN2 expression in osteoblasts.

In the present study, we also aimed to elucidate the mechanisms through which LPA influences CCN2 expression in osteoblasts. Our results suggest that LPA affects CCN2 expression in osteoblasts through the GPCR/protein kinase C (PKC) and protein kinase A (PKA) pathways.

Materials and methods

Reagents

1-Oleoyl lysophosphatidic acid (sodium salt) (LPA; item no. 62215; Cayman Chemical Co., Ann Arbor, MI, USA) and Kil16425 (S1315; Selleck Chemicals, Houston, TX, USA) were dissolved in 4 mM sterile stock solutions and stored at −20°C until use. Phorbol 12-myristate 13-acetate (PMA; S1819; PKA agonist), forskolin (S1612; PKA activator), staurosporine (S1882; PKC inhibitor) and H-89 (S1643; selective inhibitor of PKA) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). CCN2 polyclonal rabbit anti-mouse CTGF primary antibody (ab6992, diluted 1:2,000) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit anti-GAPDH primary antibody (ab181602, diluted 1:10,000) were purchased from Abcam (Cambridge, UK).

Cell cultures and cell treatment

The MC3T3-E1 cell line (China Center for Type Culture Collection, Wuhan, China) was cultured in α-modified minimal essential medium (α-MEM) containing 10% fetal bovine serum (FBS) (both from HyClone, Logan, UT, USA), 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37°C in a 5% CO2 atmosphere; the medium was changed to fresh medium at an interval of 3 days. The cells were seeded on 6-well plates or 6 cm dishes to examine gene and protein expression. After the cells reached confluence, the culture medium was replaced with α-MEM and 1% penicillin-streptomycin without FBS for 12 h. The medium was then replaced with α-MEM containing 4% bovine seum albumin (BSA) and varying doses of LPA, PMA or forskolin. Inhibitors, such as staurosporine and H-89, were used to suppress PKC and PKA activity, respectively. After co-incubation was performed for the indicated periods of time (0–12 h), the MC3T3-E1 cells were lysed for the subsequent analysis of RNA and protein expression.

Measurement of cell viability

The viability of the MC3T3-E1 cells was determined as previously described (21). Briefly, following incubation of the cells with increasing concentrations of LPA (0–40 µM) for different periods of time (12 to 72 h), cell viability was determined using a cell viability analyzer (Beckman Coulter, Fullerton, CA, USA).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

MTT assay was performed as described in a previous study (22). Briefly, the MC3T3-E1 cells were seeded in 96-well plates (3×103 cells/well) and incubated with increasing concentrations of LPA (0–40 µM). At the end of the incubation period, 20 µl of 5 mg/ml MTT were added; the cells were then further incubated for 4 h. Subsequently, the supernatant was removed, and 150 µl of dimethyl sulfoxide (DMSO) were added to each well. The absorbance of each well was detected at 490 nm. Data are presented as percentage of the control group.

RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RNA isolation and RT-qPCR were performed as previously described (23). Briefly, total RNA was isolated from the ME3T3-E1 cells in each treatment group using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was synthesized from 1 µg of total RNA using a Revert Aid First-Strand cDNA Synthesis kit (#K1622; Thermo Fisher Scientific, Waltham, MA, USA). The obtained cDNA was then amplified by quantitative PCR (qPCR) using an ABI 7900 HT Real-Time PCR system, (Applied Biosystems, Foster City, CA, USA) and SYBR®-Green Real-Time PCR Master Mix (Toyobo, Tokyo, Japan), under the following conditions: stage 1, 95°C for 10 min; stage 2, 95°C for 10 sec, 55°C for 20 sec, followed by 40 cycles of 72°C for 15 sec; stage 3, melt curve stage. GAPDH was used for the normalization of gene expression. Relative gene expression was analyzed using the 2−ΔΔCt method based on normalization to the endogenous control, GAPDH, and calculation of the threshold cycle (Ct) value difference. The results were represented as a fold change of the comparative expression level. The sequences of the forward and reverse primers were as follows: 5′-GCCTACCGACTGGAA GACACATTT-3′ and 5′-TTACGCCATGTCTCCGTACA TCTT-3′ for the CCN2 gene; 5′-ACCACAGTCCATGCCA TCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for the internal control GAPDH gene.

Protein extraction and western blot analysis

The cells were treated with the indicated stimulants (LPA, LPA + Kil16425, LPA + staurosporine, PMA, LPA + H-89, forskolin, or LPA + forskolin) for 6 h. Subsequently, the cells were then washed thrice with phosphate-buffered saline (PBS) and treated with RIPA lysis buffer. The cell lysates were cleared by centrifugation; protein concentration in the supernatant was measured by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). Total proteins (20 µg) were separated through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and the proteins were then transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% (w/v) nonfat milk and incubated overnight with the primary antibodies (CCN2, PKC or GAPDH) at the specified dilutions in TBST buffer (CW0043; Beijing Conwin Biotech Co., Beijing, China) according to the manufacturer's instructions. Protein expression was detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific). GADPH was used as an internal control protein.

PKC activity assay

PKC activity was assessed by determining the translocation of PKC from the cytosol to the membrane. After the cells were treated with the indicated stimulants for the indicated periods of time, PKC in the cytosol and in the membrane of MC3T3-E1 cells was extracted using a Mem-PER Plus Membrane Protein Extraction kit (Thermo Fisher Scientific). PKC expression in the cytosol and membrane was then determined by western blot analysis. The membrane translocation of PKC was also investigated by immunofluorescence. Briefly, the MC3T3-E1 cells were plated on cover slips and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. The cells were then washed thrice with PBS buffer, permeabilized with PBS buffer containing 0.1% Triton X-100 and blocked with 1% BSA. The cells were then incubated with PKC antibody (P5704; Sigma, St. Louis, MO, USA) for 2 h at 37°C. The cells were washed again with PBS buffer thrice and incubated with PE-labeled secondary antibody (CW0113; Beijing Conwin Biotech Co.). The fluorochrome dye, 4′,6-diamidino-2-phenylindole (DAPI), was used to visualize the cell nuclei. Images were captured using a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism 5.0 software. Data are expressed as the means ± standard error of the mean (SEM) of 3 independent experiments. A value of P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of LPA on the viability and proliferation of MC3T3-E1 cells

We initially examined the effects of LPA on the viability and proliferation of the MC3T3-E1 cells. The results of cell viability assay demonstrated that LPA did not exert cytotoxic effects on the MC3T3-E1 cells (Fig. 1A). A previous study reported that LPA induced an increase in DNA synthesis in rat osteoblasts in vitro (24). However, our results from MTT assay revealed that increasing concentrations of LPA did not affect MC3T3-E1 cell proliferation in vitro (Fig. 1B).

CCN2 expression in LPA-stimulated MC3T3-E1 cells

The MC3T3-E1 cells were stimulated with 20 µM LPA and 4% BSA for 0.5, 1, 2, 4, 6, 8 and 12 h, in order to examine the effects of LPA on CCN2 expression in osteoblasts. The CCN2 mRNA expression levels were measured by RT-qPCR. The results revealed that LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation. The mRNA expression levels of CCN2 subsequently decreased (Fig. 2A). The results from western blot analysis revealed that the CCN2 protein expression levels was similarly enhanced following 6 h of stimulation with LPA (Fig. 3D).

Ki16425 antagonizes the LPA-induced increase in CCN2 expression in osteoblasts

LPA elicits its functions through receptors on plasma membranes; MC3T3-E1 cells express LPA-specific receptors with the following relative abundance pattern: LPA1 > LPA4 > LPA2 > LPA3 (2). Thus, in this study, we wished to determine whether Kil16425, a specific inhibitor of LPA1/3 (25), is was capable of antagonizing the effects of LPA on CCN2 expression in MC3T3-E1 cells. We found that pre-treatment with Ki16425 (20 µM) significantly reduced the LPA-induced increase in the mRNA expression of CCN2 in the MC3T3-E1 cells (Fig. 2B). Kil16425 also blocked the LPA-induced increase in the protein expression of CCN2 in the MC3T3-E1 cells (Fig. 3D).

The effects of LPA on CCN2 expression may be attributed to LPA1 due to the very low expression of LPA3 (2). Thus, LPA induces an increase in CCN2 expression through the LPA1 receptor in MC3T3-E1 cells.

Effects of LPA on PKC activity

LPA receptors belong to the GPCR family (9). The LPA-induced activation of GPCR induces an increase in a variety of second messengers, such as Ca2+ in the cytoplasm of osteoblasts (2,3,8,26). Ca2+ is essential for PKC membrane translocation and activation (27). The activation PKC is manifested by the membrane translocation of PKC (28) and PKC activity may be thus enhanced by LPA. In this study, the MC3T3-E1 cells were stimulated with LPA for 15 min; PKC membrane translocation was observed by immunofluorescence staining (Fig. 3A). Western blot analysis revealed that LPA increased the ratio between membrane-derived PKC and cytosolic PKC (Fig. 3B). These results indicate that LPA induces PKC membrane translocation and enhances PKC activity in osteoblasts.

Induction of CCN2 in response to LPA requires the activation of PKC

Staurosporine and PMA, an inhibitor and an agonist of PKC, respectively, were used to determine whether PKC is involved in the effects of LPA on CCN2 expression. Staurosporine (20 nM) markedly impaired the ability of LPA to enhance CCN2 expression in the MC3T3-E1 cells, whereas PMA (1 µM) mimicked LPA and induced the expression of CCN2 (Fig. 3C and D).

Effects of PKA on LPA-induced CCN2 expression

Cyclic AMP (cAMP), another second messenger, accumulates in cells expressing LPA4,5 receptors (29,30) in the presence of LPA; the accumulation of cAMP increases PKA activity. We thus examined the role of PKA in the expression of CCN2 in LPA-treated MC3T3-E1 cells. The MC3T3-E1 cells were pre-incubated with H-89 (10 µM), a selective inhibitor of PKA, for 30 min; LPA (20 µM) was then added or the cells were treated with the PKA activator, forskolin, alone for 2 and 6 h. The mRNA and protein expression levels of CCN2 in the LPA-H-89 group were enhanced compared with those of the LPA group (Fig. 4). Pre-treatment with forskolin (10 µM) significantly decreased the mRNA and protein expression levels of of CCN2 which had been increased by LPA in the MC3T3-E1 cells. No significant effects were observed in the cells treated with forskolin alone compared to the cells also treated with LPA (Fig. 4).

Discussion

LPA is a small, bioactive phospholipid that mediates multiple cellular processes (16). Previous studies have demonstrated that LPA enhances CCN2 expression in epithelial cells, myoblasts and human renal fibroblasts (13,19,20). However, the mechanisms through which LPA influences CCN2 expression in these cells remain unclear, and it is unknown whether LPA is capable of enhancing CCN2 expression in osteoblasts. In this study, we demonstrated that LPA induced CCN2 expression in MC3T3-E1 cells. To the best of our knowledge, our study provides preliminary data on LPA-induced CCN2 expression in MC3T3-E1 cells.

LPA receptors are GPCRs expressed in MC3T3-E1 cells (2). Given the dominant expression of LPA1, we used Kil16425, a specific inhibitor of LPA1/3 (25) to identify which LPA receptor subtype is involved in the LPA-induced increase in CCN2 expression. The results revealed that the LPA-induced increase in CCN2 expression in osteoblasts was antagonized by Kil16425.

The activation of LPA receptors by LPA increases the amount of various second messengers, such as Ca2+, which are required for PKC activation and membrane translocation in osteoblasts (31). Thus, in this study, we evaluated PKC activity in LPA-stimulated MC3T3-E1 cells. The membrane translocation of PKC in the MC3T3-E1 cells was induced by LPA following 15 min of stimulation. This finding indicated that LPA enhanced PKC activity in osteoblasts.

We subsequently examined the role of PKC in the LPA-induced increase in CCN2 expression. Pre-treatment with staurosporine significantly reduced the LPA-induced increase in CCN2 expression. Treatment with PMA alone enhanced the effects of LPA on CCN2 expression. Taken together, these findings demonstrate that the PKC pathway is involved in the LPA-induced increase in CCN2 expression, and thus a positive association between CCN2 expression and PKC activity was established herein. To the best of our knowledge, the present study is the first to demonstrate that the PKC pathway is involved in the promoting effects of LPA on CCN2 expression in MC3T3-E1 cells.

The cells pre-treated with the PKA inhibitor, H-89, and subsequently stimulated with LPA exhibited higher expression levels of CCN2 than the cells stimulated with LPA alone. The PKA activator, forskolin, had no effect on CCN2 expression compared to the cells stimulated with LPA alone. Our results are similar to those of other studies which concluded that cAMP stimulated CCN2 degradation in microvessel cells and decreased CCN2 expression in human renal fibroblasts; moreover, the results demonstrated that elevated intracellular cAMP levels activated PKA which in turn prevented the induction of CCN2 (20,32). Those results suggested that the PKA pathway reduced the LPA-induced increase in CCN2 expression in MC3T3-E1 cells.

The present study revealed the mechanisms responsible for the LPA-induced increase in CCN2 expression in MC3T3-E1 cells. We identified LPA1 as an important receptor in this process. Moreover, the PKC and PKA pathways were shown to be involved in the LPA-induced increase in CCN2 expression. The hypothetical signaling pathway of the LPA-induced increase in CCN2 expression in osteoblasts is illustrated in Fig. 5. These findings elucidated the mechanisms responsible for the increase in CCN2 expression in LPA-stimulated cells.

Abbreviations:

LPA

lysophosphatidic acid

CTGF

connective tissue growth factor

GPCRs

G-protein-coupled receptors

PKC

protein kinase C

PKA

protein kinase A

PMA

phorbol 12-myristate 13-acetate

α-MEM

α-modified minimal essential medium

FBS

fetal bovine serum

DMSO

dimethyl sulfoxide

RT-qPCR

reverse transcription-quantitative polymerase chain reaction

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

PBS

phosphate-buffered saline

PFA

paraformaldehyde

cAMP

cyclic AMP

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grant nos. 81271107 and 81470718).

References

1 

Goetzl EJ: Pleiotypic mechanisms of cellular responses to biologically active lysophospholipids. Prostaglandins. 64:11–20. 2001. View Article : Google Scholar : PubMed/NCBI

2 

Masiello LM, Fotos JS, Galileo DS and Karin NJ: Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells. Bone. 39:72–82. 2006. View Article : Google Scholar : PubMed/NCBI

3 

Liu YB, Kharode Y, Bodine PV, Yaworsky PJ, Robinson JA and Billiard J: LPA induces osteoblast differentiation through interplay of two receptors: LPA1 and LPA4. J Cell Biochem. 109:794–800. 2010.PubMed/NCBI

4 

Peyruchaud O, Leblanc R and David M: Pleiotropic activity of lysophosphatidic acid in bone metastasis. Biochim Biophys Acta. 1831:99–104. 2013. View Article : Google Scholar

5 

Sims SM, Panupinthu N, Lapierre DM, Pereverzev A and Dixon SJ: Lysophosphatidic acid: a potential mediator of osteoblast-osteoclast signaling in bone. Biochim Biophys Acta. 1831:109–116. 2013. View Article : Google Scholar

6 

Hurst-Kennedy J, Boyan BD and Schwartz Z: Lysophosphatidic acid signaling promotes proliferation, differentiation, and cell survival in rat growth plate chondrocytes. Biochim Biophys Acta. 1793:836–846. 2009. View Article : Google Scholar : PubMed/NCBI

7 

Eichholtz T, Jalink K, Fahrenfort I and Moolenaar WH: The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem J. 291:677–680. 1993. View Article : Google Scholar : PubMed/NCBI

8 

Panupinthu N, Rogers JT, Zhao L, Solano-Flores LP, Possmayer F, Sims SM and Dixon SJ: P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis. J Cell Biol. 181:859–871. 2008. View Article : Google Scholar : PubMed/NCBI

9 

Noguchi K, Herr D, Mutoh T and Chun J: Lysophosphatidic acid (LPA) and its receptors. Curr Opin Pharmacol. 9:15–23. 2009. View Article : Google Scholar : PubMed/NCBI

10 

Fukushima N and Chun J: The LPA receptors. Prostaglandins Other Lipid Mediat. 64:21–32. 2001. View Article : Google Scholar : PubMed/NCBI

11 

Dziak R, Yang BM, Leung BW, Li S, Marzec N, Margarone J and Bobek L: Effects of sphingosine-1-phosphate and lysophosphatidic acid on human osteoblastic cells. Prostaglandins Leukot Essent Fatty Acids. 68:239–249. 2003. View Article : Google Scholar : PubMed/NCBI

12 

Vial C, Zúñiga LM, Cabello-Verrugio C, Cañón P, Fadic R and Brandan E: Skeletal muscle cells express the profibrotic cytokine connective tissue growth factor (CTGF/CCN2), which induces their dedifferentiation. J Cell Physiol. 215:410–421. 2008. View Article : Google Scholar

13 

Wiedmaier N, Müller S, Köberle M, Manncke B, Krejci J, Autenrieth IB and Bohn E: Bacteria induce CTGF and CYR61 expression in epithelial cells in a lysophosphatidic acid receptor-dependent manner. Int J Med Microbiol. 298:231–243. 2008. View Article : Google Scholar

14 

Brigstock DR: The CCN family: a new stimulus package. J Endocrinol. 178:169–175. 2003. View Article : Google Scholar : PubMed/NCBI

15 

Brigstock DR, Goldschmeding R, Katsube KI, Lam SC, Lau LF, Lyons K, Naus C, Perbal B, Riser B, Takigawa M and Yeger H: Proposal for a unified CCN nomenclature. Mol Pathol. 56:127–128. 2003. View Article : Google Scholar : PubMed/NCBI

16 

Kawaki H, Kubota S, Suzuki A, Yamada T, Matsumura T, Mandai T, Yao M, Maeda T, Lyons KM and Takigawa M: Functional requirement of CCN2 for intramembranous bone formation in embryonic mice. Biochem Biophys Res Commun. 366:450–456. 2008. View Article : Google Scholar

17 

Nakanishi T, Nishida T, Shimo T, Kobayashi K, Kubo T, Tamatani T, Tezuka K and Takigawa M: Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. Endocrinology. 141:264–273. 2000.

18 

Arnott JA, Lambi AG, Mundy C, Hendesi H, Pixley RA, Owen TA, Safadi FF and Popoff SN: The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis. Crit Rev Eukaryot Gene Expr. 21:43–69. 2011. View Article : Google Scholar : PubMed/NCBI

19 

Cabello-Verrugio C, Córdova G, Vial C, Zúñiga LM and Brandan E: Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway. Cell Signal. 23:449–457. 2011. View Article : Google Scholar

20 

Heusinger-Ribeiro J, Eberlein M, Wahab NA and Goppelt-Struebe M: Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP. J Am Soc Nephrol. 12:1853–1861. 2001.PubMed/NCBI

21 

Chen G, Zhu JY, Zhang ZL, Zhang W, Ren JG, Wu M, Hong ZY, Lv C, Pang DW and Zhao YF: Transformation of cell-derived microparticles into quantum-dot-labeled nanovectors for antitumor siRNA delivery. Angew Chem Int Ed Engl. 54:1036–1040. 2015. View Article : Google Scholar

22 

Li YG, Zhu W, Tao JP, Xin P, Liu MY, Li JB and Wei M: Resveratrol protects cardiomyocytes from oxidative stress through SIRT1 and mitochondrial biogenesis signaling pathways. Biochem Biophys Res Commun. 438:270–276. 2013. View Article : Google Scholar : PubMed/NCBI

23 

Liu Q, Wan Q, Yang R, Zhou H and Li Z: Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation. Biochem Biophys Res Commun. 424:182–188. 2012. View Article : Google Scholar : PubMed/NCBI

24 

Grey A, Banovic T, Naot D, Hill B, Callon K, Reid I and Cornish J: Lysophosphatidic acid is an osteoblast mitogen whose proliferative actions involve G(i) proteins and protein kinase C, but not P42/44 mitogen-activated protein kinases. Endocrinology. 142:1098–1106. 2001.PubMed/NCBI

25 

Ohta H, Sato K, Murata N, Damirin A, Malchinkhuu E, Kon J, Kimura T, Tobo M, Yamazaki Y, Watanabe T, et al: Ki16425, a subtype-selective antagonist for EDG-family lysophosphatidic acid receptors. Mol Pharmacol. 64:994–1005. 2003. View Article : Google Scholar : PubMed/NCBI

26 

Aki Y, Kondo A, Nakamura H and Togari A: Lysophosphatidic acid-stimulated interleukin-6 and -8 synthesis through LPA1 receptors on human osteoblasts. Arch Oral Biol. 53:207–213. 2008. View Article : Google Scholar

27 

Escribá PV, Wedegaertner PB, Goñi FM and Vögler O: Lipid-protein interactions in GPCR-associated signaling. Biochim Biophys Acta. 1768:836–852. 2007. View Article : Google Scholar

28 

Yuan X, Chen H, Li X, Dai M, Zeng H, Shan L, Sun Q and Zhang W: Inhibition of protein kinase C by isojacareubin suppresses hepatocellular carcinoma metastasis and induces apoptosis in vitro and in vivo. Sci Rep. 5:128892015. View Article : Google Scholar : PubMed/NCBI

29 

Lee CW, Rivera R, Gardell S, Dubin AE and Chun J: GPR92 as a new G12/13 - and Gq - coupled lysophosphatidic acid receptor that increases cAMP, LPA5. J Biol Chem. 281:23589–23597. 2006. View Article : Google Scholar : PubMed/NCBI

30 

Lee CW, Rivera R, Dubin AE and Chun J: LPA(4)/GPR23 is a lysophosphatidic acid (LPA) receptor utilizing G(s)-, G(q)/G(i)-mediated calcium signaling and G(12/13)-mediated Rho activation. J Biol Chem. 282:4310–4317. 2007. View Article : Google Scholar

31 

Lin ME, Herr DR and Chun J: Lysophosphatidic acid (LPA) receptors: signaling properties and disease relevance. Prostaglandins Other Lipid Mediat. 91:130–138. 2010. View Article : Google Scholar : PubMed/NCBI

32 

Boes M, Dake BL, Booth BA, Erondu NE, Oh Y, Hwa V, Rosenfeld R and Bar RS: Connective tissue growth factor (IGFBP-rP2) expression and regulation in cultured bovine endothelial cells. Endocrinology. 140:1575–1580. 1999.PubMed/NCBI

Related Articles

Journal Cover

February-2016
Volume 37 Issue 2

Print ISSN: 1107-3756
Online ISSN:1791-244X

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Yu Z, Li D, Huang X, Xing X, Yu R, Li Z and Li Z: Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways. Int J Mol Med 37: 468-474, 2016
APA
Yu, Z., Li, D., Huang, X., Xing, X., Yu, R., Li, Z., & Li, Z. (2016). Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways. International Journal of Molecular Medicine, 37, 468-474. https://doi.org/10.3892/ijmm.2016.2450
MLA
Yu, Z., Li, D., Huang, X., Xing, X., Yu, R., Li, Z., Li, Z."Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways". International Journal of Molecular Medicine 37.2 (2016): 468-474.
Chicago
Yu, Z., Li, D., Huang, X., Xing, X., Yu, R., Li, Z., Li, Z."Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways". International Journal of Molecular Medicine 37, no. 2 (2016): 468-474. https://doi.org/10.3892/ijmm.2016.2450