The BMP and FGF pathways reciprocally regulate odontoblast differentiation

Karo Parsegian

doi:10.1080/03008207.2022.2094789

. Author manuscript; available in PMC: 2024 Jan 1.

Published in final edited form as: Connect Tissue Res. 2022 Jul 11;64(1):53–63. doi: 10.1080/03008207.2022.2094789

The BMP and FGF pathways reciprocally regulate odontoblast differentiation

Karo Parsegian ^1,²

PMCID: PMC9832171 NIHMSID: NIHMS1820332 PMID: 35816114

Abstract

Purpose.

Previous studies demonstrated that the exposure of primary dental pulp (DP) cultures to fibroblast growth factor 2 (FGF2) between days 3-7 exerted significant and long-lasting stimulatory effects on odontoblast differentiation and Dspp expression. These effects involved the increased expression of components of bone morphogenetic protein (BMP) signaling and were reverted by a BMP inhibitor noggin. FGF2 also transiently stimulated osteoblast differentiation and the expression of Ibsp and Dmp1. The present study aimed to further explore interactions between BMP and FGF signaling during odontoblast and osteoblast differentiation in DP cultures.

Materials and Methods.

Cultures were established using DP tissue isolated from non-transgenic and fluorescent reporter (DSPP-Cerulean, BSP-GFP, and DMP1-mCherry) transgenic mice and exposed to BMP2, FGF2, SU5402 (an FGF receptor inhibitor), and noggin between days 3-7. Mineralization, gene expression, fluorescent protein expression, and odontoblast formation were examined using xylenol orange, quantitative PCR, fluorometric analysis, and immunocytochemistry, respectively.

Results.

BMP2 activated SMAD1/5/8 but not ERK1/2 signaling, whereas FGF2 exerted opposite effects. BMP2 did not affect mineralization, the expression of Ibsp and Dmp1, and the percentage of DSPP-Cerulean+ odontoblasts but significantly increased Dspp and DSPP-Cerulean. In cultures exposed to BMP2 and FGF2, respectively, both SU5402 and noggin led to long-lasting decreases in Dspp and DSPP-Cerulean and transient decreases in Dmp1 and DMP1-mCherry without affecting Ibsp and BSP-GFP.

Conclusion.

BMP2 and FGF2 exerted reciprocal stimulatory effects on odontoblast differentiation, whereas their effects on osteoblast differentiation were mediated independently. These data will further elucidate the perspectives of using BMP2 and FGF2 for dentin regeneration/repair.

Keywords: Bone Morphogenetic Protein 2, Dental pulp, Fibroblast Growth Factor 2, Mineralization, Odontoblasts

Introduction

Bone morphogenetic proteins (BMPs) are ubiquitously expressed signaling molecules that belong to the transforming growth factor β (TGF-β) superfamily of growth factors ^1-3. They mediate their biological effects through binding highly conserved transmembrane serine/threonine kinase receptors (BMPRs) ². This interaction triggers the activation of downstream intracellular pathways, including canonical (SMAD-dependent) and non-canonical (SMAD-independent) signaling ^2,3. The type of the activated signaling pathway depends on a specific BMP ligand that binds to BMPRs ^1,4. Due to their early and extensive pattern of expression, BMPs play an essential role in embryonic development and organogenesis, and their deregulated activity leads to various defects in organ formation and embryonic lethality ^1,3,5. In addition, BMP signaling is essential for primary and secondary dentinogenesis. In developing teeth, BMPs and BMPRs are expressed in polarizing and functional odontoblasts ^4,6,7, and their odontoblast-specific inactivation results in the dysregulated odontoblast differentiation, decreased dentin formation and its impaired mineralization, and the reduced expression of dentin-associated proteins, such as dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) ^6,8-10. In addition to primary and secondary dentinogenesis, BMPs, either released from the damaged dentin matrix ¹¹ or added exogenously ^12,13, are involved in the regulation of reparative dentinogenesis.

Fibroblast growth factor (FGF) signaling regulates tooth development in vivo ¹⁴ and exerts diverse effects on migration, proliferation, and differentiation of DP cells in vitro ¹⁵. Intense FGF2 expression is observed in subodontoblastic dental papilla cells, differentiating and mature odontoblasts, and dentin matrix ¹⁶. These observations along with the stage-specific expression of FGF receptor 1 (Fgfr1) and Fgfr2 ¹⁷ suggest that FGF signaling is involved in the regulation of various phases of odontoblast proliferation and differentiation.

Recent in vivo studies showed that the exposure of α-smooth muscle actin+ early odontoprogenitors to FGF2 enhanced their differentiation into mature odontoblasts and induced reparative dentinogenesis in a DP injury model ¹⁸. The exposure of primary DP cultures to FGF2 prior to induction of mineralization (between days 3-7, referred to as early and limited exposure) exerted significant and long-lasting increases in odontoblast differentiation. These increases involved rapid and marked increases in the expression of Dspp and the formation of DMP1−GFP+ functional odontoblasts ¹⁹ and were mediated through activated FGFR, ERK1/2, and BMP signaling ^19,20. Dmp1, a marker of functional and mature odontoblasts and osteocytes ²¹, and Ibsp, a marker of osteoblast differentiation ²², were transiently increased by FGF2. These observations suggest that long-lasting stimulatory effects of FGF signaling on odontoblast differentiation (evidenced by the increased Dspp expression) and its transient stimulatory effects on osteoblast differentiation (evidenced by the increased Ibsp and Dmp1 expression) in primary DP cultures could be mediated via interactions with BMP signaling. The present study was conducted to gain further insight into the role of interactions between BMP and FGF signaling in odontoblast and osteoblast differentiation in DP cultures.

Materials and Methods

2.1. Ethics statement.

All experimental protocols involving animal tissues ensured humane practices and were approved by the Institutional Animal Care and Use Committee (The University of Connecticut Health). They were performed in strict compliance with the IACUC, the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, NIH), and ARRIVE 2.0 (Animal Research: Reporting of In Vivo Experiments) guidelines.

2.2. Animals.

Immunocytochemistry, gene expression, and mineralization assays were performed using cultures established from non-transgenic CD1 mice. Transgenic mice were used to identify odontoblast and osteoblast lineage cells in heterogeneous primary DP cultures. Specifically, DSPP-Cerulean served as a marker of functional and mature odontoblasts ²¹, BSP-GFP was expressed in osteoblasts and osteocytes ²², and DMP1-mCherry was expressed in functional and mature odontoblasts and osteocytes ²¹. The animals were fed standard rodent chow and provided access to water ad libitum.

2.3. Cell cultures.

Primary DP cultures were established using DP tissue harvested from 5-7-day-old mouse pups (n = 8-12/experiment/group) as described previously ²³. On day 3, cells were cultured in a serum-free medium for two hours and then exposed to 50 ng/ml recombinant human BMP2 and 20 ng/ml low molecular weight bovine FGF2 (R&D Systems, Inc., Minneapolis, MN) until day 7. In these experiments, 4 mM HCl and 0.1% bovine serum albumin (BSA) fraction V in phosphate-buffered saline (PBS) served as respective vehicles (VHs) for BMP2 and FGF2. Inhibition of BMP/BMPR and FGF/FGFR signaling was performed by exposing the cultures to 300 ng/ml noggin (PeproTech, Rocky Hill, NJ) and 20 μM SU5402 (Santa Cruz Biotechnology, Santa Cruz, CA), respectively, between days 7-21 as described previously ²⁴. In these experiments, dimethyl sulfoxide and 0.1% BSA fraction V in PBS served as respective VHs for noggin and SU5402. BMP2, FGF2, noggin, SU5402, and their respective VHs were added to the cultures with the fresh culture medium every other day between days 3-7.

2.4. Immunocytochemistry.

Cultures were processed for immunocytochemistry using an anti-GFP antibody (Invitrogen, Grand Island, NY) to enhance the visualization signal of the DSPP-Cerulean transgene as described previously ^21,23. In brief, cultured cells were fixed with 3.7% formaldehyde in PBS for 4 min at room temperature (RT), incubated with 0.5% Triton X in PBS for 10 min at RT, blocked with 3% milk for 1 hr at RT and then incubated with anti-GFP Alexa Fluor 488-conjugated antibody (1: 1,000 dilution; Molecular Probes, Eugene, OR, USA) in 0.3% Triton X in PBS overnight at 4°C. The nuclei were stained with 1.0 μg/ml Hoechst 33342 dye (Invitrogen by ThermoFisher Scientific, Waltham, MA, USA) for 15 min at RT. After staining, coverslips were mounted using Dako fluorescent mounting medium (Dako North America, Inc., Carpinteria, CA, USA) and cultures were visualized under the Zeiss AxioObserver Z.1 microscope equipped with AxioCam MRc digital camera and using filters for GFPtpz and DAPI for the detection of GFP and Hoechst 33342, respectively. Exposure times were adjusted for optimum imaging and kept consistent for all time points of the culture.

The fluorescent intensity of the GFP signal in stained DSPP-Cerulean cultures was calculated as described previously ²³. In brief, the mean fluorescence intensity of GFP and Hoechst 33342 was measured using a multidetection monochromator microplate reader (Safire 2, Tecan, Research Triangle Park, NC, USA) at 500/540 and 343/483 nm wavelength (excitation/emission), respectively, and at a gain of 80. The entire area of each well was read at a scan density of 6×6 regions (high-sensitivity flash mode). Background fluorescence for GFP was measured using primary DP cultures established from non-transgenic littermates and processed for immunostaining at the same time point, and these values were subtracted from respective GFP measurements.

The percentage of DSPP-Cerulean-positive odontoblasts was calculated as the ratio of cells stained with anti-GFP antibody (DSPP-Cerulean-positive cells) to the total number of Hoechst-positive cells according to the established protocol ²³. In each experiment, approximately 10,000-20,000 Hoechst-positive cells were counted from 10-20 different areas of cultures.

For detection of phospho-SMAD1/5/8 (pSMAD1/5/8) and phospho-ERK1/2 (pERK1/2), cultures were processed for immunocytochemistry as described previously ^19,24. On day 3, cells were incubated in the serum-free culture medium for two hours and then in the fresh medium containing growth factors for one hour. Cells were fixed in 3.7% formaldehyde, incubated with 1:200 dilution of rabbit anti-mouse pSMAD1/5/8 or pERK1/2 antibodies (Cell Signaling, Boston, MA) overnight at +4°C and with 1:400 secondary Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Grand Island, NY) for one hour at RT. Cultures were processes for nuclei staining and visualization under the microscope as described above for DSPP-Cerulean.

2.5. Detection and quantification of mineralization and the intensity of fluorescent proteins in live DP cultures.

The detection and quantification of mineralization in live cultures were performed using xylenol orange (XO) staining as described previously ^19,24. Cultures were incubated with XO solution overnight (final concentration 20 μM), and the medium was replaced with the fresh one two hours and immediately before visualizing the cells. The mean fluorescence intensity of XO staining was measured using the microplate reader at 570/610 nm wavelength (excitation/emission) and the gain of 80. Background fluorescence for XO was measured using unstained primary DP cultures at the same time point, and these values were subtracted from respective XO measurements.

The detection and quantification of BSP-GFP and DMP1-mCherry were performed according to the established protocol ^21-23,25 and as described above. The mean fluorescence intensity of GFP and mCherry was measured using the microplate reader at 500/540 and 580/620 nm wavelength (excitation/emission), respectively, and at the gain of 80. Negative controls included primary DP cultures established from non-transgenic littermates and at the same time point, and these values were subtracted from respective GFP measurements.

2.6. RNA isolation and gene expression analysis.

Total RNA was isolated using the RNeasy Mini Kit and treated with RNase-free DNase to eliminate genomic DNA according to the manufacturer's instructions (Qiagen, Germantown, MD, USA). Isolated RNA was reverse transcribed to cDNA by Superscript II Reverse Transcriptase with Oligo(dT)_12-18 primers (Life Technologies, Grand Island, NY, USA). The expression of Ibsp (assay ID Mm01208381_g1), Dmp1 (assay ID Mm00803831_m1), Dspp (assay ID Mm00515666_m1), and Gapdh (assay ID Mm99999915_g1) was examined by quantitative PCR (qPCR) using TaqMan primers (Applied Biosystems by ThermoFisher Scientific, Foster City, CA, USA) following the 2^−ΔΔCT method as described previously ¹⁹. In brief, 9 ng of cDNA was combined with 5 μl TaqMan Universal PCR Master Mix (Applied Biosystems by ThermoFisher Scientific, Foster City, CA, USA), 2.5 μl H₂O and 0.5 μl TaqMan primers (total 10 μl). All qPCR reactions were run using 7900HT Fast Real-Time PCR System (Applied Biosystems by ThermoFisher Scientific, Foster City, CA, USA) under the following conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles with denaturation at 95°C for 15 s and extension at 60°C for 1 min. Amplification efficiency was determined using internal standard curves derived from a purified amplicon, diluted 2-fold (0.14-9.0 ng), and was close to 100% for all qPCR reactions. We defined the acceptable range of cycle threshold values representing gene expression to be between 10-35 cycles, according to the manufacturer’s recommendations (Applied Biosystems by ThermoFisher Scientific, Foster City, CA, USA).

2.7. Statistical analysis.

Statistical analysis was performed on data generated from all experiments at all time points. No randomization and blinding were performed to allocate control and experimental groups, and no confounders were controlled during the study. At least three independent experiments were performed per assay. The statistical analysis was performed by GraphPad Prism 9 software (GraphPad Software, La Jolla, CA) using one-way ANOVA analysis followed by the Holm-Šídák multiple comparisons test (for experiments involving SU5402 and noggin) or unpaired parametric t-test with Welch’s correction (for all other experiments). Results were expressed as the mean and standard deviation, and the statistical significance was detected when the multiplicity adjusted P-value was ≤ .01. The word “significant” throughout the text referred to statistical significance.

Results

The effects of BMP2 on SMAD1/5/8 and ERK1/2 signaling.

BMP2 (and BMP4 that served as a positive control) markedly increased the number of pSMAD1/5/8+ nuclei and the intensity of the GFP signal compared to control (VH-treated) cultures, whereas FGF2 did not affect them (Supplemental Figure S1(a)). Neither BMP2 nor BMP4 increased the number of pERK1/2+ nuclei and the intensity of the GFP signal compared to control cultures, whereas FGF2 markedly increased them (Supplemental Figure 1(b)).

The effects of BMP2 on mineralization and odontoblast and osteoblast differentiation.

BMP2 did not affect mineralization (Figures 1(a) and (b)) and the expression of Ibsp (Figure 1(c)) and Dmp1 (Figure 1(d)) at any time point compared to the respective controls. In contrast, it significantly increased Dspp expression at all time points (Figure 1(e)) compared to control. The effects of BMP2 on Ibsp, Dmp1, and Dspp were similar to those on respective fluorescent proteins. Specifically, it did not affect the intensity of BSP-GFP (Figures 2(a) and (b)) and DMP1-mCherry (Figures 2(a) and (c)) at any time point but significantly increased DSPP-Cerulean at all time points (Figures 2(d) and (e)) compared to respective controls. There were no significant differences in the percentage of DSPP-Cerulean+ odontoblasts between control and BMP2-treated cultures at any time point (Figure 2(f)).

Figure 2. — Cultures were exposed to VH and 50 ng/ml BMP2 between days 3-7. On days 10, 14, and 21, the fluorescent intensity of BSP-GFP, DMP1-mCherry, and DSPP-Cerulean transgenes was examined using a fluorescent plate reader, and the percentage of DSPP-Cerulean+ odontoblasts was determined using immunocytochemistry as described in Materials and Methods.

**(a)** At each time point, representative images of the same areas in live cultures were taken under BF (upper row) and epifluorescent light using the filter for detection of BSP-GFP (middle row) and DMP1-mCherry (bottom row) on days 10, 14, and 21 as described in Materials and Methods. The magnifications of all micrographs are identical. The scale bar = 100 μm.

The histograms showed changes in the intensity of BSP-GFP **(b)** and DMP1-mCherry **(c)** in VH- and BMP2-treated cultures.

**(d)** Representative images of the same areas in fixed cultures were taken under BF (upper row) and epifluorescent light using the filter for detection of GFP (middle row) and Hoechst/GFP overlaid images (bottom row) on days 10, 14, and 21 as described in Materials and Methods. GFP antibody was used to enhance the signal of the Cerulean fluorescent protein. Negative control included cultures exposed to 50 ng/ml BMP2 with the omission of the primary antibody and demonstrated no detectable signal (*not shown*).

**(e)** The histogram showed changes in the intensity of DSPP-Cerulean in VH- and BMP2-treated cultures.

**(f)** The table showed the percentages of DSPP-Cerulean+ odontoblasts in VH- and BMP2-treated cultures on days 10, 14, and 21. The percentage of DSPP-Cerulean+ odontoblasts was calculated as a ratio between Cerulean+ cells (visualized by anti-GFP antibody) and the total number of Hoechst+ cells as described previously ²³. BMP2 did not have significant effects on the percentage of DSPP-Cerulean+ odontoblasts at any time point compared to VH control (~1.1-fold).

Results of all experiments are expressed in absolute values and represent the mean ± SD of at least three independent experiments (n ≥3), each represented as a rhomboid-shaped symbol; *P ≤ .01 relative to the respective controls at each time point.

Abbreviations: BF (brightfield), SD (standard deviation), VH (vehicle).

The effects of the interaction between BMP and FGF signaling on mineralization and odontoblast and osteoblast differentiation.

FGFR inhibitor SU5402 did not alter the effects of BMP2 on mineralization on days 10 and 14 and significantly decreased it on day 21 compared to BMP2 alone (Figure 3(a) and Supplemental Figure S2). SU5402 did not affect the effects of BMP2 on Ibsp and BSP-GFP (Figures 3(b) and (e) and Supplemental Figure S3) but significantly decreased Dmp1 and DMP1-mCherry on days 14 and 21 (Figures 3(c) and (f) and Supplemental Figure S3) and Dspp and DSPP-Cerulean at all time points (Figures 3(d) and (g) and Supplemental Figure S4).

Figure 3. — Cultures were exposed to VH, 50 ng/ml BMP2, 20 μM SU5402, and BMP2 + SU5402 between days 3-7. On days 10, 14, and 21, the fluorescent intensity of XO, BSP-GFP, DMP1-mCherry, and DSPP-Cerulean transgenes was examined using a fluorescent plate reader, and the expression of *Ibsp, Dmp1*, and *Dspp* was examined by TaqMan qPCR as described in Materials and Methods.

**(a)** The histogram showed changes in the intensity of XO staining in VH- and BMP2-treated cultures.

The histograms showed changes in the expression of *Ibsp* **(b)**, *Dmp1* **(c)**, *Dspp* **(d)**, BSP-GFP **(e)**, DMP1-mCherry **(f)**, and DSPP-GFP **(g)**.

The expression of *Ibsp* and *Dmp1* was normalized to VH on day 7 (not shown on the histograms), which was set up at 1. The expression of *Dspp* was normalized to VH on day 10 (since no *Dspp* expression was detected in VH-treated control cultures on day 7), which was set up at 1. Results of all experiments are expressed in absolute values and represent the mean ± SD of at least three independent experiments (n ≥3), each represented as a rhomboid-shaped symbol; *P ≤ .01 relative to VH, ^#P ≤ .01 relative to BMP2, and °P ≤ .01 relative to SU5402.

Abbreviations: BF (brightfield), GFP (green fluorescent protein), SD (standard deviation), VH (vehicle), XO (xylenol orange).

Consistent with our previous studies ¹⁹, the early and limited exposure of DP cultures to FGF2 did not affect mineralization on days 10 and 14 but significantly increased it on day 21 compared to VH control (Figure 4(a) and Supplemental Figure S2). FGF2 did not affect Ibsp and BSP-GFP at any time point (Figures 4(b) and (e) and Supplemental Figure S3) but significantly increased Dmp1 and DMP1-mCherry on day 10 (Figures 4(c) and (f) and Supplemental Figure S3) and Dspp and DSPP-Cerulean at all time points (Figures 4(d) and (g) and Supplemental Figure S4) compared to the respective controls. The inhibition of BMP/BMPR signaling by noggin did not affect the effects of FGF2 on the extent of mineralization (Figure 4(a) and Supplemental Figure S2) and Ibsp and BSP-GFP (Figures 4(b) and (e) and Supplemental Figure S3) at any time point compared to the respective controls. Noggin significantly decreased the effects of BMP2 on Dmp1 and DMP1-mCherry (Figures 4(c) and (f) and Supplemental Figure S3) and Dspp and DSPP-Cerulean (Figures 4(d) and (g) and Supplemental Figure S4).

Figure 4. — Cultures were exposed to VH, 20 ng/ml FGF2, 300 ng/ml noggin, and combined FGF2 + noggin between days 3-7. On days 10, 14, and 21, the fluorescent intensity of XO, BSP-GFP, DMP1-mCherry, and DSPP-GFP transgenes was examined using a fluorescent plate reader, and the expression of *Ibsp, Dmp1*, and *Dspp* was examined by TaqMan qPCR as described in Materials and Methods.

**(a)** The histogram showed changes in the intensity of XO staining in VH- and FGF2-treated cultures.

The histograms showed changes in the expression of *Ibsp* **(b)**, *Dmp1* **(c)**, *Dspp* **(d),** BSP-GFP **(e)**, DMP1-mCherry **(f)**, and DSPP-GFP **(g)**.

Expression of *Ibsp* and *Dmp1* was normalized to VH on day 7 (not shown on the histograms), which was set up at 1. Expression of *Dspp* was normalized to VH on day 10 (since no *Dspp* expression was detected in VH-treated control cultures on day 7), which was set up at 1. Results of all experiments are expressed in absolute values and represent the mean ± SD of at least three independent experiments (n ≥3), each represented as a rhomboid-shaped symbol; *P ≤ .01 relative to VH, ^#P ≤ .01 relative to FGF2, and °P ≤ .01 relative to noggin.

Abbreviations: BF (brightfield), GFP (green fluorescent protein), SD (standard deviation), VH (vehicle), XO (xylenol orange).

Discussion

The present study aimed to supplement our previous findings and gain further insight into the role of interactions between BMP and FGF signaling in odontoblast and osteoblast differentiation by exposing primary DP cultures to exogenous BMP2 between days 3-7. This exposure resulted in the rapidly activated canonical (SMAD1/5/8) but not non-canonical (ERK1/2) signaling, whereas FGF2 exerted opposite effects. These results suggest that the effects of BMP2 in primary DP cultures could be mediated through the activation of SMAD1/5/8 but not ERK1/2 signaling. Previous in vitro studies demonstrated that BMP2-mediated odontoblast differentiation and Dspp expression involved rapidly activated SMAD1/5/8 signaling ^26,27. In vivo, the increased expression of BMP2 and phosphorylated SMAD1/5/8 signaling was observed in the area of reparative dentin ^28,29. Interestingly, recent in vivo studies showed that BMP-induced SMAD signaling primarily regulated the formation of coronal dentin, whereas BMP-induced non-SMAD signaling (ERK and p38) was involved in the regulation of root dentin formation ³⁰. Overall, these results suggest that BMP and FGF signaling activated a different set of signaling pathways in primary DP cultures.

The present study also demonstrated that BMP2 did not affect mineralization but led to significant and long-lasting increases in the Dspp expression. These results correlate with previous studies demonstrating stimulatory effects of BMP signaling on Dspp expression and dentin formation. The exposure of DP cells ³¹ and MD10-F2 immortalized preodontoblasts ³² to BMP2 led to increased odontoblast differentiation and Dspp expression. The inactivation of BMP signaling in Wnt1-expressing dental mesenchymal cells ⁶ or polarizing odontoblasts ^9,33 led to the markedly impaired dentin formation, its abnormal structure, and the decreased expression of Dmp1, Dspp, and transcription factors that belonged to BMP signaling (Dlx3, Dlx5, and Sp7). Although the deletion of either Bmp2 or Bmp4 in Dmp1-expressing odontoblasts did not result in dentin phenotype, double knockout of these genes led to the disorganized collagen fiber orientation, decreased DSP expression, decreased dentin thickness and density, and increased predentin thickness ⁸. In reactionary dentin, the inhibition of BMP signaling did not affect matrix mineralization but impaired its architecture ¹¹.

Although SU5402 and noggin did not impact the effects of BMP2 and FGF2, respectively, on mineralization, they significantly decreased the expression of Dspp and DSPP-Cerulean. The magnitude of these decreases was quite similar (~1.85-4.6-fold for SU5402 and ~1.7-3.1-fold for noggin) suggesting a reciprocal nature of these effects. However, the present and previous ^19,24 studies highlighted several differences in the mechanisms of stimulatory effects of BMP2 and FGF2 on odontoblast differentiation in primary DP cultures. First, BMP2-mediated increases in the Dspp expression in our study were not due to the increased formation of odontoblasts, but rather due to their increased maturity, as the percentages of DSPP-Cerulean+ odontoblasts were similar in control and BMP2-treated cultures. This mechanism of the increased odontoblast differentiation is different from the one of FGF2, which stimulated both odontoblast formation and maturity ^19,24. Second, as mentioned above, BMP2 and FGF2 activated different sets of signaling pathways; specifically, BMP2 stimulated phosphorylation of SMAD1/5/8 but not ERK1/2, whereas FGF2 stimulated phosphorylation of ERK1/2 but not SMAD1/5/8. Since BMP and FGF signaling reciprocally regulated Dspp, both SMAD and ERK1/2 signaling were likely to converge on a common downstream target that acted as a transcription factor for Dspp (Supplemental Figure S5(a)). One of the possible candidates, RUNX2, is a downstream target of both SMAD1/5/8 ^34,35 and ERK1/2 ³⁶ pathways and acts as a transcriptional regulator of Dspp ^37,38. Our qPCR analysis showed that Runx2 was significantly and rapidly increased by both FGF2 ²⁰ and BMP2 (unpublished data) and correlated with the respective increases in Dspp. The inhibition of SMAD1/5/8 by dorsomorphin only partially decreased the expression of Runx2 and Dspp, whereas the inhibition of either BMPR or ERK1/2 pathways (by noggin and siRNA, respectively) almost completely inhibited Runx2 and Dspp ³⁹.

There are several other possible crosstalk points between the BMP and FGF signaling pathways in addition to RUNX2 (Supplemental Figure S5(b)). Previous studies have shown that FGF2-mediated increases in Dspp correlated with the increased expression of Fgfr1c, which was significantly increased in the population of 2.3-GFP+ cells committed to the odontoblast lineage ¹⁹. Studies using neural-crest-derived PC12 cells have shown that BMP2 could synergistically stimulate the differentiation of these cells by modulating the expression of FGFR1 via SMAD signaling ⁴⁰. We have previously shown that stimulatory effects of FGF2 on Dspp and Fgfr1 correlated with the increased expression of Bmp2, which were decreased by SU5402, U0126, and noggin ¹⁹. BMP2 protein may be then released into the extracellular space where it acts in an autocrine-paracrine manner through binding to the transmembrane BMPRs on the target cells. The upregulation of Bmp2 by FGF2 in DP cultures could also involve the downregulation of noggin, as shown in other cell types ^41-43. Finally, the crosstalk between BMP and FGF signaling could occur at the level of SMAD/ERK interaction. Previous studies have shown that the overexpression of thymosin β-4 in human DP cultures led to the increased levels of Bmp2, Bmp4, Runx2, SMAD1/5/8, and ERK1/2, which all were partially downregulated by noggin ⁴⁴. These results suggest that at least to some extent, the interaction between BMP and FGF signaling could occur at the SMAD/ERK level. Overall, these results suggest that BMP and FGF could crosstalk at the level of downstream targets (such as SMAD/ERK and/or RUNX2), growth factor ligands, and their receptors; these interactions are downregulated or inhibited by specific inhibitors, such as SU5402, U0126, and noggin. The elucidation of the specific mechanisms underlying the interaction between BMP and FGF signaling would address the current limitation of the present work.

Contrary to the reciprocal effects of BMP2 and FGF2 on odontoblast differentiation, their regulation of osteoblast differentiation in primary DP cultures appears to be independent of each other (evidenced by similar levels of Ibsp and BSP-GFP). The effects of SU5402 and noggin on Dmp1 and DMP1-mCherry likely reflect their effects on odontoblasts, as these effects correlated with those on Dspp. These events could be due to a different set of signaling pathways and/or transcriptional factors that BMP2 and FGF2 activate (Supplemental Figure S6). Since the crosstalk between BMP and FGF signaling has been demonstrated in bone and other tissues ⁴⁵, our results may highlight differences between the BMP and FGF signaling pathways in the differentiation of osteoprogenitors residing in the DP as opposed to other tissues (such as bone and bone marrow). This is consistent with previous observations on the differences in the effects of FGF signaling on osteoblast differentiation in primary DP vs. bone marrow cultures ²³.

Overall, the results of the present and previous studies provide further insight into the use of BMP2 and FGF2 to achieve more predictable outcomes of dentin regeneration/repair protocols. In the case of FGF2, only the early exposure increased odontoblast differentiation/maturation ¹⁹, whereas the late ²⁴ and continuous ²³ exposures inhibited it. Therefore, dentin regeneration/repair protocols should avoid the exposure of differentiating DP cells to FGF2. In the case of BMP2, both early and late exposures increased odontoblast differentiation. These results highlight the complexity of the effects of growth factors in the heterogeneous cellular environment that contains cells of various lineages and at various stages of maturity. Clinicians should consider these differences to achieve more predictable outcomes of dentin regeneration/repair protocols.

Supplementary Material

Supp 1

Supplemental Figure S1. The effects of BMP2 on the phosphorylation of SMAD1/5/8 and ERK1/2 proteins in primary DP cultures. Cultures were established as described in the Materials and Methods. On day 3, cultures were serum-deprived for 5 hrs and then exposed to VH, 50 ng/ml BMP2, 50 ng/ml BMP4, and 20 ng/ml FGF2 for 1 hr and processed for immunocytochemistry using anti-SMAD1/5/8 and anti-ERK1/2 antibodies as described previously ^19,24. (a) Representative images of the same areas in fixed cultures were taken under BF (upper row) and epifluorescent light using a filter for GFPtpz to visualize pSMAD1/5/8 (pSMAD, middle row). The bottom row represents overlaid images of pSMAD1/5/8 with Hoechst 33342 used to visualize nuclei (pSMAD1/5/8/Hoechst). Negative control included primary DP cultures exposed to 50 ng/ml BMP4 with the omission of the primary antibody (Neg. control). The magnifications of all micrographs are identical. The scale bar = 100 μm. (b) Representative images of the same areas in fixed cultures were taken under BF (upper row) and epifluorescent light using the filter for GFPtpz to visualize pERK1/2 (pERK1/2, middle row). The bottom row represents overlaid images of pERK1/2 protein with Hoechst 33342 used to visualize nuclei (pERK1/2/Hoechst). Negative control included primary DP cultures exposed to 20 ng/ml FGF2 with the omission of the primary antibody (Neg. control). The magnifications of all micrographs are identical. The scale bar = 200 μm. Abbreviations: BF (brightfield), VH (vehicle).

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Supp 2

Supplemental Figure S2. Effects of the inhibition of FGF and BMP signaling on the extent of mineralization in primary DP cultures. Cultures were prepared and exposed to BMP2, FGF2, SU5402 (the FGF/FGFR inhibitor), BMP2 + SU5402, noggin (the BMP/BMPR inhibitor), and FGF2 + noggin between days 3-7 as described in the Materials and Methods and shown in Figures 3 and 4. At each time point, representative images of the same areas in live cultures were taken under BF (upper row) and epifluorescent light using a TRITC filter for detection of XO (bottom row). The magnifications of all micrographs are identical. The scale bar = 100 μm. Abbreviations: BF (brightfield), TRITC (tetramethylrhodamine), VH (vehicle), XO (xylenol orange).

NIHMS1820332-supplement-Supp_2.jpg^{(641.2KB, jpg)}

Supp 3

Supplemental Figure S3. Effects of the inhibition of FGF and BMP signaling on the expression of BSP-GFP and DMP1-mCherry fluorescent proteins in primary DP cultures. Cultures were prepared and exposed to BMP2, FGF2, SU5402 (the FGF/FGFR inhibitor), BMP2 + SU5402, noggin (the BMP/BMPR inhibitor), and FGF2 + noggin between days 3-7 as described in the Materials and Methods and shown in Figures 3 and 4 and Supplemental Figure S2. At each time point, representative images of the same areas in live cultures were taken under BF (upper row) and epifluorescent light using filters for detection of BSP-GFP (middle row) and DMP1-mCherry (bottom row) on days 10, 14, and 21 as described in Materials and Methods. The magnifications of all micrographs are identical. The scale bar = 100 μm. Abbreviations: BF (brightfield), GFP (green fluorescent protein), VH (vehicle).

NIHMS1820332-supplement-Supp_3.jpg^{(1MB, jpg)}

Supp 4

Supplemental Figure S4. Effects of the inhibition of FGF and BMP signaling on the expression of DSPP-Cerulean fluorescent protein in primary DP cultures. Cultures were prepared and treated with SU5402 (the FGF/FGFR inhibitor) and noggin (the BMP/BMPR inhibitor) between days 3-7 as described in the Materials and Methods and shown in Figures 3 and 4 and Supplemental Figures S2 and S3. At each time point, representative images of the same area in live DP cultures were taken under BF (upper row) and epifluorescent light using the filter for detection of GFP (middle row) as described in the Materials and Methods. The bottom row represents overlaid images of GFP with Hoechst 33342 (Hoechst/GFP) detected using a DAPI filter. At all time points, negative control included primary DP cultures processed for immunostaining on day 21 with the omission of the primary antibody (Neg. control). The magnifications of all micrographs are identical. The scale bar = 100 μm. Abbreviations: BF (brightfield), DAPI (4′,6-diamidino-2-phenylindole), GFP (green fluorescent protein), VH (vehicle).

NIHMS1820332-supplement-Supp_4.jpg^{(723.4KB, jpg)}

Supp 5

Supplemental Figure S5. The schematic representation of the proposed interaction between the BMP and FGF signaling pathways in the regulation of odontoblast differentiation in primary DP cultures. (a) The binding of BMP2 to BMPR triggers the activation/phosphorylation of SMAD1/5/8 signaling. The binding of FGF2 to FGFR triggers the activation/phosphorylation of ERK1/2 signaling. Both SMAD and ERK signaling converge on a common downstream mediator, such as RUNX2, that enters the nucleus, binds to the promoter region of Dspp, and stimulates its expression and odontoblast differentiation. The inhibition of BMPR/SMAD, FGFR, and ERK1/2 by noggin, SU5402, and U0126, respectively, will inhibit the transcriptional activation of Dspp by RUNX2. In addition, both BMP and FGF signaling pathways could possibly crosstalk at the level of SMAD/ERK. (b) The SMAD and ERK signaling triggered by BMPR and FGFR, respectively, activate a downstream transcription factor (TF) that stimulates the expression of BMP and FGF ligands as well as BMP and FGF receptors and subsequent formation of the respective proteins. These proteins are released from the cell where they can regulate BMPR and FGFR signaling.

NIHMS1820332-supplement-Supp_5.tiff^{(18.9MB, tiff)}

Supp 6

Supplemental Figure S6. The schematic representation of the proposed interaction between the BMP and FGF signaling pathways in the regulation of osteoblast differentiation in primary DP cultures. The activation of BMPR/SMAD1/5/8 and FGFR/ERK1/2 signaling leads to the activation of different transcription factors (TF 1 and TF 2, respectively) that enter the nucleus and regulate the expression of Ibsp and Dmp1.

NIHMS1820332-supplement-Supp_6.tiff^{(11MB, tiff)}

ACKNOWLEDGMENTS

The author would like to thank Mrs. Barbara Rogers and Drs. Ivo Kalajzic and Peter Maye for providing reagents, valuable input, and technical assistance in various aspects of this study.

FUNDING

This work was supported by the NIH under grants R01-DE016689 and T90-DE022526 to Dr. Mina Mina (University of Connecticut Health, Farmington, CT, USA).

Footnotes

DECLARATION OF INTERESTS

The author denies any conflicts of interest related to this study.

REFERENCES

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Associated Data

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Supplementary Materials

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[R1] 1.Bragdon B, Moseychuk O, Saldanha S, King D, Julian J, Nohe A. Bone morphogenetic proteins: a critical review. Cell Signal. Apr 2011;23(4):609–20. doi: 10.1016/j.cellsig.2010.10.003 [DOI] [PubMed] [Google Scholar]

[R2] 2.Katagiri T, Watabe T. Bone Morphogenetic Proteins. Cold Spring Harb Perspect Biol. Jun 1 2016;8(6)doi: 10.1101/cshperspect.a021899 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Wu M, Chen G, Li YP. TGF-beta and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res. 2016;4:16009. doi: 10.1038/boneres.2016.9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Malik Z, Roth DM, Eaton F, Theodor JM, Graf D. Mesenchymal Bmp7 Controls Onset of Tooth Mineralization: A Novel Way to Regulate Molar Cusp Shape. Front Physiol. 2020;11:698. doi: 10.3389/fphys.2020.00698 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Wang RN, et al. Bone Morphogenetic Protein (BMP) signaling in development and human diseases. Genes Dis. Sep 2014;1(1):87–105. doi: 10.1016/j.gendis.2014.07.005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Malik Z, Alexiou M, Hallgrimsson B, Economides AN, Luder HU, Graf D. Bone Morphogenetic Protein 2 Coordinates Early Tooth Mineralization. J Dent Res. Feb 1 2018:22034518758044. doi: 10.1177/0022034518758044 [DOI] [PubMed] [Google Scholar]

[R7] 7.Nadiri A, Kuchler-Bopp S, Perrin-Schmitt F, Lesot H. Expression patterns of BMPRs in the developing mouse molar. Cell Tissue Res. Apr 2006;324(1):33–40. doi: 10.1007/s00441-005-0120-1 [DOI] [PubMed] [Google Scholar]

[R8] 8.Jani P, Liu C, Zhang H, Younes K, Benson MD, Qin C. The role of bone morphogenetic proteins 2 and 4 in mouse dentinogenesis. Arch Oral Biol. Jun 2018;90:33–39. doi: 10.1016/j.archoralbio.2018.02.004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Yang W, Harris MA, Cui Y, Mishina Y, Harris SE, Gluhak-Heinrich J. Bmp2 is required for odontoblast differentiation and pulp vasculogenesis. Research Support, N.I.H., Extramural. J Dent Res. Jan 2012;91(1):58–64. doi: 10.1177/0022034511424409 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Yuan G, Yang G, Zheng Y, Zhu X, Chen Z, Zhang Z, Chen Y. The non-canonical BMP and Wnt/beta-catenin signaling pathways orchestrate early tooth development. Development. Jan 01 2015;142(1):128–39. doi: 10.1242/dev.117887 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Neves VCM, Sharpe PT. Regulation of Reactionary Dentine Formation. J Dent Res. Apr 2018;97(4):416–422. doi: 10.1177/0022034517743431 [DOI] [PubMed] [Google Scholar]

[R12] 12.Goldberg M, Six N, Decup F, Buch D, Soheili Majd E, Lasfargues JJ, Salih E, Stanislawski L. Application of bioactive molecules in pulp-capping situations. Comparative Study Research Support, Non-U.S. Gov't. Adv Dent Res. Aug 2001;15:91–5. [DOI] [PubMed] [Google Scholar]

[R13] 13.Six N, Lasfargues JJ, Goldberg M. Differential repair responses in the coronal and radicular areas of the exposed rat molar pulp induced by recombinant human bone morphogenetic protein 7 (osteogenic protein 1). Comparative Study Research Support, Non-U.S. Gov't. Arch Oral Biol. Mar 2002;47(3):177–87. [DOI] [PubMed] [Google Scholar]

[R14] 14.Li CY, Prochazka J, Goodwin AF, Klein OD. Fibroblast growth factor signaling in mammalian tooth development. Odontology. Jan 2014;102(1):1–13. doi: 10.1007/s10266-013-0142-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Vaseenon S, Chattipakorn N, Chattipakorn SC. The possible role of basic fibroblast growth factor in dental pulp. Arch Oral Biol. Jan 2020;109:104574. doi: 10.1016/j.archoralbio.2019.104574 [DOI] [PubMed] [Google Scholar]

[R16] 16.Russo LG, Maharajan P, Maharajan V. Basic fibroblast growth factor (FGF-2) in mouse tooth morphogenesis. Growth factors. 1998;15(2):125–33. [DOI] [PubMed] [Google Scholar]

[R17] 17.Kettunen P, Karavanova I, Thesleff I. Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, −2, −3, and of FGFR4; and stimulation of cell proliferation by FGF-2, −4, −8, and −9. Research Support, Non-U.S. Gov't. Dev Genet. 1998;22(4):374–85. doi: 10.1002/(SICI)1520-6408(1998)22:4<374::AID-DVG7>3.0.CO;2-3 [DOI] [PubMed] [Google Scholar]

[R18] 18.Vidovic-Zdrilic I, Vining KH, Vijaykumar A, Kalajzic I, Mooney DJ, Mina M. FGF2 Enhances Odontoblast Differentiation by αSMA(+) Progenitors In Vivo. J Dent Res. Sep 2018;97(10):1170–1177. doi: 10.1177/0022034518769827 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Sagomonyants K, Kalajzic I, Maye P, Mina M. Enhanced Dentinogenesis of Pulp Progenitors by Early Exposure to FGF2. J Dent Res. Nov 2015;94(11):1582–90. doi: 10.1177/0022034515599768 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Sagomonyants K, Mina M. Biphasic effects of FGF2 on odontoblast differentiation involve changes in the BMP and Wnt signaling pathways. Connect Tissue Res. Aug 2014;55 Suppl 1:53–6. doi: 10.3109/03008207.2014.923867 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Vijaykumar A, Ghassem-Zadeh S, Vidovic-Zdrilic I, Komitas K, Adameyko I, Krivanek J, Fu Y, Maye P, Mina M. Generation and characterization of DSPP-Cerulean/DMP1-Cherry reporter mice. Genesis. Jul 4 2019:e23324. doi: 10.1002/dvg.23324 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Vijaykumar A, Dyrkacz P, Vidovic-Zdrilic I, Maye P, Mina M. Expression of BSP-GFPtpz Transgene during Osteogenesis and Reparative Dentinogenesis. J Dent Res. Jan 2020;99(1):89–97. doi: 10.1177/0022034519885089 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Sagomonyants K, Mina M. Stage-specific effects of fibroblast growth factor 2 on the differentiation of dental pulp cells. Cells Tissues Organs. 2014;199(5-6):311–28. doi: 10.1159/000371343 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Sagomonyants K, Kalajzic I, Maye P, Mina M. FGF Signaling Prevents the Terminal Differentiation of Odontoblasts. J Dent Res. Jun 2017;96(6):663–670. doi: 10.1177/0022034517691732 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Maye P, Stover ML, Liu Y, Rowe DW, Gong S, Lichtler AC. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs. BMC Biotechnol. Mar 13 2009;9:20. doi: 10.1186/1472-6750-9-20 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Cho YD, Yoon WJ, Woo KM, Baek JH, Park JC, Ryoo HM. The canonical BMP signaling pathway plays a crucial part in stimulation of dentin sialophosphoprotein expression by BMP-2. J Biol Chem. Nov 19 2010;285(47):36369–76. doi:M110.103093 [pii] 10.1074/jbc.M110.103093 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

The BMP and FGF pathways reciprocally regulate odontoblast differentiation

Karo Parsegian