Aberrant BMP2 Signaling in Patients Diagnosed with Osteoporosis

Abstract

The most common bone disease in humans is osteoporosis (OP). Current therapeutics targeting OP have several negative side effects. Bone morphogenetic protein 2 (BMP2) is a potent growth factor that is known to activate both osteoblasts and osteoclasts. It completes these actions through both SMAD-dependent and SMAD-independent signaling. A novel interaction between the BMP type Ia receptor (BMPRIa) and casein kinase II (CK2) was discovered, and several CK2 phosphorylation sites were identified. A corresponding blocking peptide (named CK2.3) was designed to further elucidate the phosphorylation site's function. Previously, CK2.3 demonstrated an increased osteoblast activity and decreased osteoclast activity in a variety of animal models, cell lines, and isolated human osteoblasts. It is hypothesized that CK2.3 completes these actions through the BMP signaling pathway. Furthermore, it was recently discovered that BMP2 did not elicit an osteogenic response in osteoblasts from patients diagnosed with OP, while CK2.3 did. In this study, we explore where in the BMP pathway the signaling disparity or defect lies in those diagnosed with OP. We found that osteoblasts isolated from patients diagnosed with OP did not activate SMAD or ERK signaling after BMP2 stimulation. When OP osteoblasts were stimulated with BMP2, both BMPRIa and CK2 expression significantly decreased. This indicates a major disparity within the BMP signaling pathway in patients diagnosed with osteoporosis.

Keywords: BMP2; BMPRIa; CK2; CK2.3; osteoporosis.

Figure 1

Mature osteoblasts were extracted from osteoporosis (OP) patients and stimulated with BMP2, CK2.3 or were left unstimulated (US) for five days. On the fifth day cells were fixed and fluorescently stained for pERK (green) and the nucleus (blue). (a) CK2.3 significantly increased fluorescent intensity when compared to BMP2 and US cells. BMP2 stimulation significantly decreased fluorescent intensity when compared to CK2.3 stimulated cells. (b) Lysates were also collected from extracted osteoblasts from OP patients and run on an SDS-Page gel to separate the proteins. The separated proteins were then transferred onto an immunoblot and pERK and β-actin protein levels were detected. Expression was detected through densiometric analysis using ImageJ. CK2.3 significantly increased expression of pERK when compared with US and BMP2 stimulated cells. Error bars represent standard error of the mean (SEM). “a” denotes statistically significant to US, “b” denotes statistically significant to BMP2 stimulated cells, and “c” denotes statistically significant to CK2.3 stimulated cells (p < 0.05).

Figure 1

Mature osteoblasts were extracted from osteoporosis (OP) patients and stimulated with BMP2, CK2.3 or were left unstimulated (US) for five days. On the fifth day cells were fixed and fluorescently stained for pERK (green) and the nucleus (blue). (a) CK2.3 significantly increased fluorescent intensity when compared to BMP2 and US cells. BMP2 stimulation significantly decreased fluorescent intensity when compared to CK2.3 stimulated cells. (b) Lysates were also collected from extracted osteoblasts from OP patients and run on an SDS-Page gel to separate the proteins. The separated proteins were then transferred onto an immunoblot and pERK and β-actin protein levels were detected. Expression was detected through densiometric analysis using ImageJ. CK2.3 significantly increased expression of pERK when compared with US and BMP2 stimulated cells. Error bars represent standard error of the mean (SEM). “a” denotes statistically significant to US, “b” denotes statistically significant to BMP2 stimulated cells, and “c” denotes statistically significant to CK2.3 stimulated cells (p < 0.05).

Figure 2

Mature osteoblasts were extracted from OP patients and stimulated with BMP2, CK2.3 or were left US for five days. On the fifth day, cells were fixed and fluorescently stained for pSMAD (green) and the nucleus (blue). (a) pSMAD fluorescent intensity remains unchanged under all stimulations, while BMP2 stimulation indicates a slight decrease in expression, this was not significant. (b) Lysates were also collected from extract osteoblasts from OP patients and run on an SDS-Page gel to separate the proteins. The separated proteins were then transferred onto an immunoblot and pSMAD and β-actin protein levels were detected. Expression was detected through densiometric analysis. BMP2 stimulation seems to decrease pSMAD expression, but this difference was not significant. Error bars represent standard error of the mean (SEM). (p < 0.05).

Figure 3

(a) A schematic representing how femoral heads were sliced down the midsagittal plane, and what region of interest was used for both the methyl methacrylate (MMA) experiments and the explant experiments. (b) Diagram showing the explant experimental set up. The trabecular bone fragment was removed from the femoral head, washed with PBS incubated with antibiotics/antimycotics for 10 min. The fragments were placed in a six well plate (one fragment per well) with DMEM. They were stimulated as designated for five days, following which they were either stained for cell viability and imaged or they were fixed with 4.4% PFA, immunofluorescently stained and then imaged. (c) Cell viability was assessed through a Calcein and Hoescht stain, and viable cells were counted. Under all stimulations and conditions cells were 80% or more viable within the explants. Error bars represent standard error of the mean (SEM).

Figure 4

Explants from OP and control patients were prepared as previously described and stimulated with BMP2, CK2.3, or left US. After the fifth day the explants were fixed and stained for OC (green), ALP (red), and the nucleus (blue). (a) Control and OP explants stimulated with CK2.3 significantly increased expression of OC when compared to US and BMP2 stimulation. OP explants stimulated with BMP2 significantly decreased expression of OC. (b) Representative 2D images depicting the nuclear stain overlayed with the OC stain. (c) Both control and OP explants stimulated with CK2.3 significantly increased expression of ALP when compared to control and BMP2 stimulation. BMP2 stimulation significantly decreased ALP expression in both control and OP explants. (d) Representative 2D images depicting the nuclear stain overlaid with the ALP stain. Error bars represent standard error of the mean (SEM). “a” denotes statistically significant to control US explants, “b” denotes statistically significant to control BMP2 stimulated explants, “c” denotes statistically significant to control CK2.3 stimulated explants, “d” denotes statistically significant to OP US explants, “e” denotes statistically significant to OP BMP2 stimulated explants, and “f” denotes statistically significant to OP CK2.3 stimulated explants (p < 0.05).

Figure 5

Female OP and OA (control) trabecular bone slices were embedded in MMA and fluorescently stained for BMPRIa (green), CK2α (red), and the nucleus (blue). OP BMPRIa and CK2α expression was significantly higher than the expression levels in control bone slices. Error bars represent standard error of the mean (SEM). “a” denotes statistically significant to control BMPRIa levels, “b” denotes statistically significant control CK2α levels (p < 0.05).

Figure 6

Trabecular bone slices were removed from isolated femoral heads. OP and control explants were prepared as previously described. Fixed explants were stained fluorescently for BMPRIa (green), CK2α (red), and the nucleus (blue). (a) In control explants CK2.3 and BMP2 significantly increased BMPRIa expression when compared to US explants. (b) In OP explants, BMP2 significantly decreased BMPRIa expression when compared to CK2.3 and US explants. (c) In control explants, BMP2 and CK2.3 significantly increased expression of CK2α when compared to US explants. (d) In OP explants, BMP2 stimulation significantly decreased expression of CK2α when compared to US and CK2.3 stimulated explants. Error bars represent standard error of the mean (SEM). “a” denotes statistically significant to US explants, “b” denotes statistically significant to BMP2 stimulated explants, and “c” denotes statistically significant to CK2.3 stimulated explants (p < 0.05).

Figure 7

Osteoblasts extracted from OP patients were stimulated with BMP2, CK2.3, or left US for five days. After the fifth day, the cells were fixed and stained for BMPRIa (green), CK2α (red), and the nucleus (blue). (a) CK2.3 and US cells significantly increased expression of BMPRIa when compared to BMP2 stimulation. (b) BMP2 stimulation significantly decreased expression of CK2α when compared to CK2.3 and US cells. (c) RNA was extracted from OP patients’ osteoblasts after they were stimulated with BMP2, CK2.3, or left US for five days. BMP2 significantly decreased BMPRIa gene expression when compared to US and CK2.3 stimulated cells. Error bars represent standard error of the mean (SEM). “a” denotes statistically significant to US, “b” denotes statistically significant to BMP2, and “c” denotes statistically significant to CK2.3 (p < 0.05).

Figure 8

Proposed mechanism of BMP2 (a) and CK2.3 (b) signaling in POP primary osteoblasts. (a) Upon treatment with BMP2, POP primary osteoblasts are unresponsive and the downstream signaling pathways, ERK and SMAD, are not activated and osteogenesis is decreased. (b) When stimulated with CK2.3, this peptide is up-taken by POP primary osteoblasts and subsequently, the ERK and SMAD signaling pathways are activated independently of BMP2, which increases osteogenesis.