Volume 48, Issue 6, Pages 1409-1416 (June 2011) Phosphate increases bone morphogenetic protein-2 expression through cAMP- dependent protein kinase and ERK1/2 pathways in human dental pulp cells  Hiroyuki Tada, Eiji Nemoto, Brian L. Foster, Martha J. Somerman, Hidetoshi Shimauchi  Bone  Volume 48, Issue 6, Pages 1409-1416 (June 2011) DOI: 10.1016/j.bone.2011.03.675 Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 1 Elevated extracellular Pi stimulates BMP-2 expression in human dental pulp cells. A confluent monolayer of cells was cultured in medium containing 5% FBS with 0.01μM dexamethasone and 50μg/ml ascorbic acid for 9days (A), with the indicated concentration of Pi for 48h (B and F), with 3mM Pi for the indicated times (C), or with 3mM Pi for 48h in the presence of 10μg/ml CHX after pretreatment with CHX for 45min (E). Confluent monolayer of human PDL cells (two donors) or C3H10T1/2 were cultured in medium containing 5% FBS with 3mM Pi for 48h (G and H). Total cellular RNA was extracted at the indicated time, and transcripts were analyzed by conventional or real-time RT-PCR. (D): A sub-confluent monolayer of cells was transiently transfected with hBMP2 promoter plasmid and cultured in medium containing 5% FBS with 3mM Pi for 36h. Luciferase activities were measured in cell lysates and normalized to that of a Renilla transfection control. (A): Data are representative of two independent experiments. Fragment sizes are 189bp (DSPP), 155bp (DMP-1), and 156bp (GAPDH). (B–H): Representative data of three separate experiments are shown as means±SD of triplicate assays. Statistical significance is indicated (*, P<0.05 compared with untreated control; §, P<0.05 compared with respective control). Bone 2011 48, 1409-1416DOI: (10.1016/j.bone.2011.03.675) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 2 Pi-mediated BMP-2 expression does not require activation of the PKC pathway. (A): Pulp cells, at monolayer confluence, were pretreated with 0.1 or 1μM GF-109203X or 1μM U73122 for 30min followed by stimulation in medium containing 5% FBS with 3mM Pi for 48h in the presence of inhibitor. (B): Pulp cells, at monolayer confluence, were pretreated with 1μM GF-109203X for 30min followed by stimulation in medium containing 5% FBS with 10nM PMA for 6h in the presence of inhibitor. All samples, including the control, were adjusted to contain 0.1% (v/v) DMSO in the media during cell culture. Total cellular RNA was extracted and transcripts were analyzed by real-time PCR. Representative data of three separate experiments are shown as means±SD of triplicate assays. Statistical significance is indicated (*, P<0.05 compared with untreated control; §, P<0.05 compared with 10nM PMA-treated control). Bone 2011 48, 1409-1416DOI: (10.1016/j.bone.2011.03.675) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 3 Pi-mediated BMP-2 expression requires the intracellular cAMP and PKA pathways. (A): Pulp cells, at confluence, were pretreated with 20μM MDL-12,330A or 20μM H89 for 30min followed by stimulation with 3mM Pi for 48h in the presence of inhibitor in medium containing 5% FBS. Total cellular RNA was extracted and transcripts were analyzed by real-time PCR. (B): Pulp cells, at confluence, were preincubated with 500μM phosphodiesterase inhibitor, IBMX for 10min, followed by stimulation with 3mM Pi for the indicated times in the presence of inhibitor in medium containing 5% FBS. The amount of cAMP in the lysate was analyzed using a cAMP enzyme immunoassay as described in Materials and methods. (C): Pulp cells, at confluence, were pretreated with 500μM 8-Br-cAMP for 15min followed by stimulation with 3mM Pi for 48h in the presence of 8-Br-cAMP in medium containing 5% FBS. Total cellular RNA was extracted and transcripts were analyzed by real-time PCR. All samples, including the control, were adjusted to contain 0.1% (v/v) DMSO in the media during cell culture. Representative data of three separate experiments are shown as means±SD of triplicate assays. Statistical significance is indicated (*, P<0.05 compared with untreated control; §, P<0.05 compared with respective control). Bone 2011 48, 1409-1416DOI: (10.1016/j.bone.2011.03.675) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 4 Pi-mediated BMP-2 expression requires activation of the ERK1/2 pathway, which operates independently of cAMP-dependent signaling pathway. (A): Pulp cells, at confluence, were stimulated with 3mM Pi for the indicated time in medium containing 1% FBS. Cell lysates were analyzed by Western blotting with an anti-phospho-ERK1/2 antibody to detect the phosphorylation of ERK1/2. An antibody against total ERK1/2 was used as a control. Findings are representative of three independent experiments. (B): The expression levels of phosphorylated ERK1 in (A) were quantified via densitometry scanning using NIH Image software. Relative expression levels of phosphorylated ERK1 were normalized to ERK1. (C): Pulp cells, at confluence, were pretreated with 20μM PD98059 for 30min followed by stimulation with 3mM Pi for 48h in the presence of inhibitor in medium containing 5% FBS. Total cellular RNA was extracted and transcripts were analyzed by real-time PCR. Representative data of three separate experiments are shown as means±SD of triplicate assays. Statistical significance is indicated (*, P<0.05 compared with untreated control; §, P<0.05 compared with respective control). (D): A confluent monolayer of cells was pretreated with 20μM MDL-12,330A for 30min followed by stimulation with 3mM Pi for the indicated times in the presence of inhibitor in medium containing 1% FBS. Cell lysates were analyzed by Western blotting using anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. Findings are representative of three independent experiments. (E): The relative expression levels of phosphorylated ERK1, normalized to ERK1, were quantified via densitometry scanning using NIH Image software. (A–E): All samples, including the control, were adjusted to contain 0.1% (v/v) DMSO in the media during cell culture. Bone 2011 48, 1409-1416DOI: (10.1016/j.bone.2011.03.675) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 5 Human dental pulp cells express the NaPi-III type transporters (PIT1 and PIT2), but not NaPi-I, -IIa, -IIb, and -IIc transporters. (A): A template cDNA from the human multi-tissue cDNA library (kidney) was used as control. (B): Expression of PIT1, PIT2, NaPi-I, NaPi-IIa, NaPi-IIb, and NaPi-IIc mRNA by human dental pulp cells. Total cellular RNA was extracted from cells, at confluence, obtained from two different donors, and transcripts were analyzed by RT-PCR. Data are representative of two independent experiments. Fragment sizes are 150bp (PIT1), 167bp (PIT2), 154bp (NaPi-I), 191bp (NaPi-IIa), 157bp (NaPi-IIb), 167bp (NaPi-IIc), and 156bp (GAPDH). (C and D): A confluent monolayer of cells was cultured in medium containing 5% FBS in the presence or absence of 3mM Pi for the indicated time. Total cellular RNA was extracted and transcripts were analyzed by real-time PCR. Representative data of three separate experiments are shown as means±SD of triplicate assays. Statistical significance is indicated (*, P<0.05 compared with untreated control; §, P<0.05 compared with respective control). Bone 2011 48, 1409-1416DOI: (10.1016/j.bone.2011.03.675) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 6 PFA and PPi inhibit Pi-mediated BMP-2 increase. (A and B): A confluent monolayer of pulp cells was pretreated with 300μM PFA or the indicated concentrations of PPi for 30min followed by stimulation with 3mM Pi for 48h in the presence of the designated inhibitor. Transcripts of BMP-2 were analyzed by real-time PCR. (C): A confluent monolayer of cells was pretreated with PFA as (A) followed by stimulation with 3mM Pi or 25μM simvastatin for 72h in medium containing 5% FBS in the presence of PFA. The amount of BMP-2 in the supernatant was analyzed by ELISA. Representative data of three separate experiments are shown as means±SD of triplicate assays (A–C). Statistical significance is indicated (*, P<0.05 compared with untreated control; §, P<0.05 compared with respective control). Bone 2011 48, 1409-1416DOI: (10.1016/j.bone.2011.03.675) Copyright © 2011 Elsevier Inc. Terms and Conditions