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.

Immunomodulatory Effects of Pentoxifylline: Profiling Data Based on RAW 264.7 Cellular Signaling

“…In the IP-HPLC, housekeeping proteins, standard β-actin, α-tubulin, and GAPDH, were used as internal controls. Expression changes of housekeeping proteins were adjusted to ≤±5% using a proportional baseline algorithm [34]. PTX seemed to suppress proliferation-related proteins, up-regulated cMyc/MAX signaling in 12 and 24 h samples; however, decreased in the 48 h sample and is thought to promote cellular proliferation up to 24 h and suppress it more.…”

4-hexylresorcinol-induced protein expression changes in human umbilical cord vein endothelial cells as determined by immunoprecipitation high-performance liquid chromatography

“…The ratios of the protein levels between the experimental and control groups were calculated and plotted. Protein expressional changes of less than ±5%, ±5-10%, ±10-20%, or over ±20% changes were described as minimal, slight, significant, or marked, respectively [19,[30][31][32].…”

“…Using precision UV spectroscopy, the ratio of the protein level can be determined by comparing it with a control protein level. Although the basic principle is similar to that of an enzyme-linked immunosorbent assay, IP-HPLC is a rapid, accurate, and reproducible method [25][26][27]32].…”

Pentoxifylline-induced Protein Expression Change in RAW 264.7 Cells as Determined by Immunoprecipitation-based High Performance Liquid Chromatography

“…The ratios of the protein levels between the experimental and control groups were plotted into line and star graphs. Protein expressional changes of less than ±5%, ±5-10%, ±10-20%, or over ±20% changes were described as minimal, slight, significant, or marked, respectively [30][31][32][33]40]. The housekeeping proteins including β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were simultaneously used as internal controls.…”

“…In the previous study, the IP-HPLC results were compared with western blot data using cytoplasmic housekeeping protein (β-actin), the former showed minute error ranges less than ± 5% which were appropriate for statistical analysis, while the latter showed a large error range of more than 20% which were impossible to be analyzed statistically [40] (see Supplementary data 2). Therefore, the present study mainly performed IP-HPLC, and its results were compared to representative findings of ICC and western blot performed with some selected antisera, even though ICC and western blot are usually involved with great error range (≥20%).…”