Extensive protein expression changes induced by pamidronate in RAW 264.7 cells as determined by IP-HPLC

Abstract

Background: Bisphosphonate therapy has become a popular treatment for osteoporosis, Paget's disease, multiple myeloma, osteogenesis imperfecta, myocardial infarction, and cancer despite its serious side effects. Bisphosphonate-induced molecular signaling changes in cells are still not clearly elucidated.

Methods: As bisphosphonates are primarily engulfed by macrophages, we treated RAW 264.7 cells (a murine macrophage cell line) with pamidronate and investigated global protein expressional changes in cells by immunoprecipitation high performance liquid chromatography (IP-HPLC) using 218 antisera.

Results: Pamidronate upregulated proliferation-activating proteins associated with p53/Rb/E2F and Wnt/β-catenin pathways, but downregulated the downstream of RAS signaling, pAKT1/2/3, ERK-1, and p-ERK-1, and subsequently suppressed cMyc/MAX/MAD network. However, in situ proliferation index of pamidronate-treated RAW264.7 cells was slightly increased by 3.2% vs. non-treated controls. Pamidronate-treated cells showed increase in the expressions of histone- and DNA methylation-related proteins but decrease of protein translation-related proteins. NFkB signaling was also suppressed as indicated by the down-regulations of p38 and p-p38 and the up-regulation of mTOR, while the protein expressions related to cellular protection, HSP-70, NRF2, JNK-1, and LC3 were upregulated. Consequently, pamidronate downregulated the protein expressions related to immediate inflammation,cellular differentiation, survival, angiogenesis, and osteoclastogenesis, but upregulated PARP-1 and FAS-mediated apoptosis proteins. These observations suggest pamidronate affects global protein expressions in RAW 264.7 cells by stimulating cellular proliferation, protection, and apoptosis but suppressing immediate inflammation, differentiation, osteoclastogenesis, and angiogenesis. Accordingly, pamidronate appears to affect macrophages in several ways eliciting not only its therapeutic effects but also atypical epigenetic modification, protein translation, RAS and NFkB signalings. Therefore, our observations suggest pamidronate-induced protein expressions are dynamic, and the affected proteins should be monitored by IP-HPLC to achieve the therapeutic goals during treatment.

Keywords: Bisphosphonate; Global protein expressions; IP-HPLC; Molecular signaling; Pamidronate; RAW 264.7 cells.

Figure 1. In situ proliferation assay of RAW 264.7 cells.

Increases in cell numbers were determined by counting on Petri dishes (A–F), and proliferation indices (%) were calculated by expressing cell growths (final-initial cell counts) as percentages of initial cells counts. Pamidronate-treated (6.5 μm) RAW 264.7 cells had a slightly higher mean proliferation index (73.1 ± 2.32% at 24 h and 74.7 ± 2.8% at 48 h) than non-treated controls (69.9 ± 2.46%) (G) PB: pamidronate.

Figure 2. Expressions of proliferation-related proteins, cMyc/MAX/MAD network proteins, p53/Rb/E2F signaling proteins, and Wnt/β-catenin signaling proteins.

Expressions of proliferation-related proteins (A and B), cMyc/MAX/MAD network proteins (C or D), p53/Rb/E2F signaling proteins (E and F), and Wnt/β-catenin signaling proteins (F or H) in pamidronate-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs (A), (C), (E) and (G) show protein expressional changes on the same scale (%) vs. culture time (12, 24, or 48 h), whereas the star plots (B, D, F and H) show the differential expression levels of proteins after 12, 24, or 48 h of treatment on appropriate scales (%). Standard error (s).

Figure 3. Expressions of epigenetic modification-related proteins, protein translation-related proteins, growth factors, and RAS signaling proteins.

Expressions of epigenetic modification-related proteins (A and B), protein translation-related proteins (C or D), growth factors (E and F), and RAS signaling proteins (G or H) in pamidronate-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs (A), (C), (E), and (G) show protein expressional changes on the same scale (%) vs. culture time (12, 24, or 48 h), whereas the star plots (B, D, F, and H) show the differential expression levels of proteins after 12, 24, or 48 h of treatment on appropriate scales (%). Standard error (s).

Figure 4. Expressions of NFkB signaling proteins, inflammatory proteins were upregulated, and inflammatory proteins downregulated.

Expressions of NFkB signaling proteins (A and B), inflammatory proteins were upregulated (C or D), and inflammatory proteins downregulated (E and F) in pamidronate treated RAW 264.7 cells as determined by IP-HPLC. Line graphs (A), (C) and (E) show protein expressional changes on the same scale (%) vs. culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of proteins after 12, 24, or 48 h of treatment on appropriate scales (%). Standard error (s).

Figure 5. Expressions of p53-mediated apoptosis-related proteins, FAS-mediated apoptosis-related proteins, and cell survival-related proteins.

Expressions of p53-mediated apoptosis-related proteins (A and B), FAS-mediated apoptosis-related proteins (C or D), and cell survival-related proteins (E and F) in RAW 264.7 cells treated with pamidronate for different times as determined by IP-HPLC. Line graphs (A), (C), and (E) show protein expressional changes on the same scale (%) vs. culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of proteins after 12, 24, or 48 h of treatment on appropriate scales (%). Standard error (s).

Figure 6. Expressions of cell protection-related proteins, differentiation-related proteins,and oncogenesis-related proteins.

Expressions of cell protection-related proteins (A and B), differentiation-related proteins (C or D), and oncogenesis-related proteins (E and F) in pamidronate-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs (A), (C) and (E) show protein expressional changes on the same scale (%) vs. culture time (12, 24, or 48 h), whereas the star plots (B, D and F) show the differential expression levels of proteins after 12, 24, or 48 h of treatment on appropriate scales (%). Standard error (s).

Figure 7. Expressions of angiogenesis-related proteins and of osteogenesis-related proteins.

Expressions of angiogenesis-related proteins (A and B) and of osteogenesis-related proteins (C or D) in pamidronate-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs (A) and (C) show protein expressional changes on the same scale (%) vs. culture time (12, 24, or 48 h), whereas the star plots (B and D) show the differential expression levels of proteins after 12, 24, or 48 h of treatment on appropriate scales (%). Standard error (s).

Figure 8. Star plot of global protein expression in pamidronate-treated RAW 264.7 cells.

Star plot of global protein expression in pamidronate-treated RAW 264.7 cells. Representative proteins (n = 73) of each signaling pathway are plotted in a circular manner. The expressions of proliferation, some growth factors, cellular apoptosis, protection, and differentiation-related proteins were upregulated, while the expressions of protein translation-, cell survival-, angiogenesis-, and osteogenesis-related proteins were downregulated. RAS signaling and NFkB signaling were suppressed by the up-regulations of the downstream effector proteins, ERK-1 (p-ERK-1) and p38 (p-p38), respectively. The expressions of inflammatory proteins and oncogenesis-related proteins in RAW 264.7 cells were variably altered, but epigenetic methylation was increased by pamidronate treatment. Blue, yellow, and red spots indicate after 12, 24, and 48 h of pamidronate treatment, respectively.

Figure 9. Highly up- and down-regulated proteins by pamidronate in RAW 264.7 cells.

The cells were reactive to pamidronate by marked upregulation of some proteins for cellular proliferation, protection, differentiation, RAS signaling, NFkB signaling, and oncogenic proteins, but gradually degenerated by marked downregulation of M2 macrophage differentiation-related inflammatory proteins and survival-related proteins and by marked upregulation of apoptosis-related proteins. The major protein expressions for angiogenesis and osteoclastogenesis were dramatically suppressed (A–C). Blue, yellow and red spots indicate after 12, 24 and 48 h of pamidronate treatment, respectively.