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

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.


INTRODUCTION
Bone undergoes constant remodeling maintained by a balance between osteoblasts and osteoclasts. Bisphosphonates inhibit the digestion of bone by causing osteoclasts to undergo apoptosis (Ito et al., 2001) and impair osteoclasts' ability to form a ruffled border (Sato et al., 1991), to adhere to the bone surface, and to synthesize protons necessary for bone resorption. Furthermore, bisphosphonates suppress osteoclast activity by decreasing osteoclast progenitor development and recruitment (Cecchini et al., 1987;Endo et al., 1993). These diphosphate analogs inhibit intermediate enzymes of mevalonate pathway and are used to treat osteoporosis and Paget's disease (historically osteitis deformans) (Abelson, 2008). In osteoporosis and Paget's disease, IV zoledronic acid is the first-choice treatment for Paget disease because of its efficacy and ease of administration (Wat, 2014). The choice of zoledronic acid as the initial agent for most patients with active Paget disease is consistent with both the 2014 clinical practice guidelines of the Endocrine Society and the 2019 Paget's Association guidelines (Singer et al., 2014).
Bisphosphonates bind calcium and are readily deposited in bone. They also change bone ultrastructures, for example, they obliterate Haversian canals and deposit irregular and thick reversal lines (Acevedo et al., 2015;Carmagnola et al., 2013;Kim et al., 2017c;Lee, 2013). The common side effects of bisphosphonates include bone pain, low blood calcium levels, nausea, and dizziness. In addition, bisphosphonate-related osteonecrosis of the jaw (BRONJ) may develop in patients who have used bisphosphonates long term (Marx et al., 2005;Ruggiero et al., 2004). Total 37 BRONJ cases out of 1,014 patients using bisphosphonates for osteoporosis treatment showed 62.6% were associated with intravenous and 37.4% with oral application (Hansen et al., 2013). The incidence of BRONJ is known to be low among patients treated with oral bisphosphonates (Sarasquete et al., 2009). The estimated prevalence of oral BRONJ was 0.05-0.07%. And the average oral bisphosphonate treatment duration was 43.1 months (range, 5-120 months) (Hong et al., 2010). Among the 320 osteoporotic patients who underwent tooth extraction, 11 developed BRONJ, reflecting an incidence rate of 3.44%. And the incidence of BRONJ increased with age, was greater in the mandible than the maxilla, and was associated with a duration of administration of more than 3 years (Jeong et al., 2017;Marx et al., 2005;Ruggiero et al., 2004). The pathophysiology of BRONJ is currently unclear. BRONJ has been attributed to infection (Chirappapha et al., 2017;Choi et al., 2017;Park et al., 2009), bisphosphonate-related osteonecrosis (Guimaraes et al., 2013), quantitative reduction of the vascularization (Kun-Darbois et al., 2018), local immune dysfunction (Hoefert et al., 2016b), genetic predisposition like polymorphisms on CYP2C8 gene (Sarasquete et al., 2009), etc. In addition, to the anti-osteoporotic effect of bisphosphonates, adjunctive bisphosphonate therapy appears to be effective at managing periodontitis (Akram et al., 2017), fibrous dysplasia (Majoor et al., 2017), and Gorham-Stout disease (Hammer et al., 2005;Kim et al., 2015). Therefore, it is believed bisphosphonates may have several systemic effects such as anti-inflammatory, anti-proliferative, and antiangiogenesis effects (Kamel, Geronikaki & Abdou, 2012;Ohlrich et al., 2016;Ribatti et al., 2008;Ribatti et al., 2008). However, the biological effects of bisphosphonates in different cells have not been clearly elucidated at the molecular level.
Pamidronate (pamidronate disodium or pamidronate disodium pentahydrate) is a nitrogen-containing bisphosphonate and used to prevent bone loss due to steroid use like glucocorticoid-induced low bone mineral density in children (Jayasena, Atapattu & Lekamwasam, 2015) or to inhibit calcium release from bone by impairing osteoclast-mediated bone resorption (Miyazaki et al., 2011), pamidronate is frequently used to treat high calcium levels (Polyzos et al., 2011). In addition, it has also been used as an experimental treatment for osteogenesis imperfecta and been studied for the treatment of complex regional pain syndrome (Chevreau et al., 2017).
Immunoprecipitation high-performance liquid chromatography (IP-HPLC) had been used previously by several authors to detect organic compounds including peptides quantitatively, but the technique used was complicated and of limited applicability (Clarke et al., 1998;Luo et al., 2013). Recently, a new IP-HPLC protocol was developed to determine protein expression levels in different biological fluids, such as blood serum, urine, saliva (Kim & Lee, 2015), inflammatory exudates (Kim et al., 2017a(Kim et al., , 2017b(Kim et al., , 2018, and different protein extracts from cells (Kim et al., 2019;Yoon et al., 2018b), liver (Yoon et al., 2018a), and cancer tissues (Kim et al., 2017d). The IP-HPLC is comparable to enzyme-linked immunosorbent assay (ELISA). The former uses protein A/G agarose beads in buffer solution and UV spectroscopy to determine protein concentrations, whereas the latter uses fluorescence-conjugated antibodies fixed in plastic wells and fluoroscopy. Furthermore, multiple trials have shown that IP-HPLC can be used to rapidly determine multiple protein levels accurately (<±5% standard deviation) and reproducibly. In the previous study (Yoon et al., 2018b), 64 proteins were assessed by IP-HPLC 4-8 times repeatedly and their results showed low error range <±5% standard deviation (shown in the raw data sheets of Supplemental Dataset 5).
When pamidronate is injected into blood vessels, it immediately chelates Ca ++ (Ebetino et al., 2011;Fernandez, Vega & Goeta, 2002) and is bound to serum albumin (90% of tiludronate) (Sansom, Necciari & Thiercelin, 1995), and subsequently recognized by macrophages, which suggests its various pharmacologic effects may be associated with the cellular functions of pamidronate-laden macrophages. Therefore, the present in vitro study was undertaken to investigate the effects of pamidronate on protein expressions in RAW 264.7 macrophages by IP-HPLC.

MATERIALS AND METHODS
RAW264.7 cell culture with pamidronate treatment RAW 264.7 cells, an immortalized murine macrophage cell line (ATCC, Manassas, VA, USA), were cultured as previously described (Yoon et al., 2018b). About 70% confluent RAW 264.7 cells grown on Petri dish surfaces were treated with 6.5 mm disodium pamidronate (similar to the therapeutic dose, 1.5 mg/kg) (Sigma, Chicago, IL, USA) for 12, 24, or 48 h; control cells were treated with 1 mL of normal saline. Cultured cells were harvested with protein lysis buffer (PRO-PREP TM ; iNtRON Biotechnology INC., Gyeonggi-do, South Korea) and immediately preserved at −70 C until required.
In situ proliferation index of RAW 264.7 cells after 24 h of pamidronate treatment RAW 264.7 cell proliferations were directly observed on plastic surfaces of Petri dishes after treatment with pamidronate at 6.5 mm for 12, 24, or 48 h, and compared with non-treated controls. When cells formed clusters containing 20-30 cells after 24 h of pamidronate treatment, ten representative histological images (taken at areas photographed before pamidronate treatment) were obtained using an inverted microscope (DP-73; Olympus Co., Tokyo, Japan). Cell numbers were obtained using the iSolution Lite program (IMT i-Solution Inc., Vancouver, BC, Canada), proliferation indices were calculated by dividing increases in cell numbers after 24 h and 48 h of culture by initial cell numbers and compared between pamidronate treatment groups and non-treated controls.
equipped with a reverse phase column and a micro-analytical detector system (SG Highteco, Seoul, South Korea), using 0.15 M NaCl/20% acetonitrile solution at 0.4 mL/min for 30 min, and proteins were detected using a UV spectrometer at 280 nm. Control and experimental samples were run sequentially to allow comparisons. For IP-HPLC, whole protein peak areas (mAU Ã s) were mathematically calculated with analytical algorithm (see Supplemental Data 1) by subtracting negative control antibody peak areas, and protein expression levels (mAU) were compared and normalized using the square roots of protein peak areas. Analyses were repeated two to six times to achieve mean standard deviations of ≤±5% (RAW data, Supplemental Data 2). Objective protein expression level (%) between experiment and control groups were calculated and results were analyzed using the standard error of the mean (Kim et al., 2019;Yoon et al., 2018aYoon et al., , 2018b. The housekeeping proteins normal β-actin, a-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were also used as internal controls. Expressional changes of housekeeping proteins were adjusted to <±5% using a proportional basal line algorithm. Protein expressional changes of ≤±5%, ±5-10%, ±10-20%, and ≥±20% change were defined as minimal, slight, meaningful, or marked, respectively.
When the IP-HPLC results were compared with the western blot data of cytoplasmic housekeeping protein (β-actin), the former exhibiting minute error ranges less than ±5% and could be analyzed statistically, while the latter showed a large error range of more than 20%, and thus it was almost impossible to analyze them statistically (see Supplemental Data 3). Therefore, the present study utilized IP-HPLC to statically analyze global protein expression changes in pamidronate-treated RAW 264.7 cells rather than Western blot method (Seo et al., 2019).

Statistical analysis
Proportional data (%) of experimental and control groups were plotted, and analyses were repeated two to six times until standard deviations were ≤±5%. Results were analyzed by measuring standard error (s ¼ AE ffiffiffiffiffiffiffiffiffiffi s 2 =n p ). The expressions of control housekeeping proteins, that is, β-actin, a-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) non-responsive (≤5%) to 12, 24, or 48 h of pamidronate treatment.

RESULTS
In situ proliferation index of RAW 264.7 cells after 24 h of pamidronate treatment Both 6.5 mm pamidronate-treated RAW 264.7 cells and non-treated controls proliferated on Petri dishes and formed large cell clusters after 24 h of culture (Figs. 1A-1F). The in situ proliferation index of pamidronate-treated RAW 264.7 cells was 73.1 ± 2.32% at 24 h, 74.7 ± 2.8% at 48 h, and that of non-treated RAW 264.7 cells was 69.9 ± 2.46% by the in situ proliferation assay (Fig. 1G). These results indicate pamidronate slightly elevated mitosis of RAW 264.7 cells, murine macrophages, by 3.2% in 24 h and 4.8% in 48 h of culture.

Effects of pamidronate on the expressions of cMyc/MAX/MAD network proteins in RAW 264.7 cells
The expressions of cMyc and MAX decreased by 12.6% and 7.9%, respectively, after 12 h of pamidronate treatment and consistently decreased by 7.4% and 1.8%, respectively, at 48 h vs. non-treated controls, whereas MAD-1 expression decreased by a maximum of 15.7% after 12 h of treatment and slightly increased by 1.7% at 48 h. On the other hand, p27 expression increased by 18.7% after 48 h of treatment (Figs. 2C and 2D). These results 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 mm) 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.   Effects of pamidronate on the expressions of translation-related proteins in RAW 264.7 cells RAW 264.7 cells treated with pamidronate showed gradual reductions in protein translation-related protein levels vs. non-treated controls. Although deoxyhypusine hydroxylase (DOHH) expression slightly increased by 17% and 5.4% after 24 and 48 h of treatment, respectively, deoxyhypusine synthase (DHS) expression was consistently reduced by 18.8% and 16.8%, respectively, at these times. The protein expressions of objective factors of protein translation, that is, eukaryotic translation initiation factor 5A-1 (eIF5A-1) and eIF5A-2, were also reduced by 2.9% and 3.2% at 48 h, respectively, while that of eukaryotic translation initiation factor 2-a kinase 3 (eIF2AK3; an inactivator of eIF2) was increased by 6.8% at 24 h (Figs. 3C and 3D). We considered that the pamidronate-induced reductions in the expressions of translation-related proteins might cause global inactivation of cellular signaling. However, changes in the levels of these protein levels which are normally abundant in cells tended to remain at <±15% after 48 h of pamidronate treatment.
Effects of pamidronate on the expressions of FAS-mediated apoptosis-related proteins in RAW 264.7 cells RAW 264.7 cells treated with pamidronate showed increases in the expressions of FAS-mediated apoptosis-related proteins as compared with non-treated controls. After treatment with pamidronate for 48 h, the expressions of death receptors on cell surfaces, that is, of FAS, FAS ligand (FASL), and FAS-associated protein with death domain (FADD), were increased by 4.6%, 15.3%, and 24.4%, respectively, and those of caspase 8, caspase 3, and c-caspase 3 were also increased by 30.8%, 27.5%, and 14.6%, respectively. On the other hand, the expressions of FLICE-like inhibitory protein (FLIP) and BH3 interacting-domain death agonist (BID) were minimally changed (<±5%) (Figs. 5C and 5D). These findings indicate pamidronate might induce apoptosis via caspase 8 and 3 through FASL/FAS/FADD signaling in RAW 264.7 cells.
Effects of pamidronate on the expressions of cell survival-related proteins in RAW 264.7 cells RAW 264.7 cells treated with pamidronate showed variable changes in the expressions of cell survival-related proteins as compared with non-treated controls. The expressions of PTEN, telomerase reverse transcriptase (TERT), NRF2, PGC-1a, PKC, p-PKC, and focal adhesion kinase (FAK) were increased by 11.2%, 8.6%, 12.1%, 10.4%, 10%, 14%, and 13.7%, respectively, after 48 h of pamidronate treatment, while those of pAKT1/2/3, survivin, BCL2, p38, p-p38, and SP-1 were reduced by 9.1%, 10.9%, 12.9%, 10.2%, 10.2%, and 9.6%, respectively. On the other hand, the expressions of SP-3, AMPK, and ATF6 hardly changed (<±5%) (Figs. 5E and 5F). These results suggested cell survival was enhanced by the up-regulations of NRF2/PGC-1a and PKC/FAK signaling, which are features of mitochondrial biogenesis and the signal transduction cascade, respectively, but reduced by the down-regulations of AKT/survivin/BCL2 and p38/SP-1 signalings, which are features of cell exposure to stressors, such as oxidative damage. These results suggest that pamidronate increases energy metabolism and signal transduction in RAW 264.7 cells, but that the abilities of their cells to overcome different cytological stressors is relatively poor.
Although RAW 264.7 cells treated with pamidronate appeared to be silent, as they exhibited reduced NFkB signaling and had low levels of antioxidant-related proteins, SOD-1, NOS1, and HO-1 in their cytoplasms, they had higher levels of the cell protection-related proteins, HSP-70, PLC-β2, PI3K, PGC-1a, JNK-1, PKC, p-PKC, FAK, and mucin 1 and 4 than non-treated controls. These observations suggest the expressions of cellular protection-related proteins, such as those involved in detoxification and autophagy, are upregulated by pamidronate in RAW 264.7 cells despite reduced RAS and NFkB signalings.
Among the angiogenesis-related proteins, the expressions of the blood vessel-forming proteins, angiogenin, VEGF-A, VEGFR2, vWF, and CMG2 were markedly reduced by pamidronate, while those of the lymphatic vessel-forming proteins, VEGF-C and LYVE-1 tended to increase slightly (<5%). Pamidronate also reduced the expressions of the extracellular matrix proteins, FGF-1, FGF-2, MMP-2, and MMP-10, which are required for de novo angiogenesis and wound healing. These results suggest pamidronate significantly suppresses the expressions of angiogenesis-related proteins in RAW 264.7 cells, and that it might be able to potently inhibit blood vessel formation in vivo.
The expressions of the major osteoblast differentiation proteins; OPG, osteocalcin, and RUNX2, and of the osteoclast differentiation proteins; RANKL, HSP-90, and cathepsin K, were markedly reduced by 48 h of pamidronate treatment, whereas the expressions of the bone matrix proteins, osteopontin, BMP-2, BMP-4, osteonectin, and ALP tended to increase. In particular, the expressions of BMP-3 (an antagonist to other BMP's in the differentiation of osteogenic progenitors) and TGF-β1 (an inhibitor of osteoclast activity) were markedly increased by pamidronate treatment. These results suggest pamidronatetreated RAW 264.7 cells are hardly differentiated into osteoclasts and give sparse influence on adjacent osteoblastic cells by expression of bone matrix proteins.

Global protein expressions in pamidronate-induced RAW 264.7 cells
Global protein expression changes of representative proteins (n = 73) from above 19 different protein signaling pathways are illustrated as a star plot in Fig. 8. Although pamidronate is low molecular weight entity, it was found to widely affect the expressions of proteins in different signaling pathways in RAW 264.7 cells. In particular, pamidronate inactivated epigenetic modification and protein translation and subsequently down-regulated the expressions of some proteins required for the proliferation, differentiation, protection, and survival of RAW 264.7 cells. The increases observed in the expressions of proliferation-related proteins were presumably related to the up-regulations of p53/Rb/E2F and Wnt/β-catenin signaling by pamidronate albeit suppression of cMyc/MAX/MAD network signaling. The suppression of RAS signaling induced by pAKT1/2/3, ERK-1, and p-ERK-1 down-regulations was followed by cMyc/MAX/MAD network down-regulation and by a subsequent inhibition in RAW 264.7 cell proliferation. Furthermore, the marked suppression of NFkB signaling appeared to be associated with elevation of PARP-1-and FAS-mediated apoptosis and reduction of cellular differentiation, survival, immediate inflammatory reaction, and wound repair.

DISCUSSION
Pamidronate is a nitrogen-containing, synthetic bisphosphonate, and its phosphate groups are believed to interfere with phosphorylation processes or interact with proteins in cells (Chen et al., 2012;Nishida et al., 2003;Stefanucci, Marrone & Agamennone, 2015). Pamidronate is not sequestered as a waste material but relatively well adapted in cells, and thus, it is presumed pamidronate is maintained as a metabolite and influences not only the intracellular mevalonate pathway and protein isoprenylation but also signaling molecules and genetic materials (Henneman et al., 2011;Iguchi et al., 2010;Kaiser et al., 2013;Tatsuda et al., 2010). It has been shown pamidronate has considerable impact on cells such as macrophages, osteoclasts, and endothelial cells, and that its long-time usage is associated with the risk of BRONJ (Hoefert et al., 2015;Sharma et al., 2016;Zhang et al., 2013). In the present study, we assessed the effects of a therapeutic dose of pamidronate on the expressions of proteins in RAW 264.7 cells by IP-HPLC. As RAW 264.7 cells are derived from murine macrophages, and their immunological roles to dialyzed coffee extract were assessed by IP-HPLC (Yoon et al., 2018b), and this study also explored RAW 264.7 cells for their macrophage roles to pamidronate.
Pamidronate-induced proliferation of RAW 264.7 cells was examined by counting cell numbers directly on Petri dishes, and protein expressional changes were determined by IP-HPLC. The in situ proliferation index of pamidronate-treated RAW 264.7 cells over 24 h was 73.1 ± 2.32%, whereas that of non-treated cells was 69.9 ± 2.46%, thus the pamidronate-induced increase was 3.2%. Furthermore, this increase in in situ proliferation index matched the pamidronate-induced increases in the expressions of different proliferation-related proteins as determined by IP-HPLC. These data suggest pamidronate can slightly activate mitosis of murine macrophages, RAW 264.7 cells.
When we explored cellular mechanism responsible for altering protein expressions in RAW 264.7 cells, we noticed that the epigenetic environment was generally inactivated by pamidronate due to the up-regulations of DMNT1, MBD4, and DMAP1 and the down-regulation of KDM3D, which would tend to increase histone and DNA methylation levels. Protein translation was also inactivated by a marked reduction in DHS expression and an increase in eIF2AK3 (an inactivator of eIF2) expression vs. non-treated controls. We suggest the concurrent inactivations of epigenetic modification and protein translation by pamidronate may have reduced global RAW 264.7 cell activity.
Pamidronate-treated RAW 264.7 cells showed a marked reduction in cMyc/MAX/ MAD network signaling during culture, and this was followed by the up-regulation of p27 (a negative regulator of G1 progression) by 16.7% at 48 h. Whereas p53/Rb/E2F signaling was enhanced by the up-regulations of p53, Rb-1, and CDK4 resulted in an increase in the expression of the objective transcription factor, E2F-1. Also, Wnt/β-catenin signaling was also enhanced by the up-regulations of Wnt-1, β-catenin, and snail, which led to the up-regulation of the objective transcription factor, TCF-1. As a result, the expressions of the proliferation-activating proteins Ki-67, PCNA, MPM2, CDK4, cyclin D2, and lamin A/C, were increased by pamidronate, and concurrently the expressions of the proliferation-inhibiting proteins p14, p15/16, p21, and p27 were compensatory increased during 48 h of pamidronate treatment. These results indicate pamidronate-treated RAW 264.7 cells were partly activated and proliferative due to increased p53/Rb/E2F and Wnt/β-catenin signaling despite a marked reduction in cMyc/MAX/MAD network signaling.
Although the expressions of NFkB, NRF2, PGC-1a, PTEN, and mTOR tended to increase (<10%) after pamidronate treatment, the expressions of p38, p-p38, GADD45, GADD153, ATF6, MDR, and SRC-1 were reduced after 48 h of treatment. In addition, the expressions of the reactive oxygen and nitrogen species-related proteins SOD-1, NOS1, and HO-1 were consistently reduced by pamidronate. These observations indicate NFkB signaling was reduced due to pamidronate-induced suppression of the downstream effector protein p38 (p-p38) in RAW 264.7 cells, and that treated cells were less reactive to oxidative or endoplasmic reticulum stress than non-treated controls.
Although pamidronate suppressed RAS and NFkB signalings simultaneously, RAW 264.7 cells expressed higher levels of the protection-related proteins HSP-70, JNK-1, PLC-β2, LC3, and FAK, the cell survival-related proteins TERT, NRF2, PGC-1a, p-PKC, and FAK, and the oncogenesis-related proteins CEA, 14-3-3, and DMBT1 than non-treated controls. In particular, increases in the expressions of HSP-70 (protects against thermal and oxidative stress), JNK-1 (a mitogen-activated protein kinase responsible to different stress stimuli), LC3 (autophagosome biogenesis protein), NRF2 (transcription factor for many antioxidant genes), 14-3-3 (a regulator of diverse signaling proteins), DMBT1 (a glycoprotein containing multiple cysteine-rich domains that interact with tumor cells), and TERT (an RNA-dependent polymerase that lengthens telomeres in DNA strands) indicated pamidronate stressed RAW 264.7 cells and stimulated them to respond by expressing protection-and oncogenesis-related proteins.
Macrophages constitute a component of the front line of host defense and mediate innate immune responses by triggering; the productions of cytokines, chemokines, and cytotoxic molecules, the mobilizations of cells such as neutrophils and other leukocytes, the phagocytosis of pathogens and their delivery to lysosomes for degradation, and the induction of autophagy (Zhang et al., 2016). Many authors have reported macrophage functions are reduced after pamidronate treatment in vitro and in vivo (Escudero & Mandalunis, 2012;Hoefert et al., 2015;Hoefert et al., 2016a;Mian et al., 1994). In the present study, although the general cytodifferentiation proteins, p63, vimentin, PLC-β2, PI3K, PKC, FAK, integrin a5, SHH, and S-100 were upregulated by pamidronate, the M2 macrophage differentiation-related proteins, TNFa, lysozyme, cathepsin G, cathepsin K, M-CSF, ICAM-1, and a1-antitrypsin were consistently downregulated, which suggested pamidronate prevented the differentiation of RAW 264.7 cells into active M2 macrophages, and resulted retarded wound healing after pamidronate treatment in vivo (Ariza Jimenez et al., 2018;Chen, Cheng & Feng, 2018).
Pamidronate-treated RAW 264.7 cells also showed increases in the expressions of the apoptosis executor proteins, caspase 8, caspase 3, and c-caspase 3, which are activated by the FAS-mediated apoptosis signaling cascade, and that the expressions of caspase 9 and c-caspase 9 were also increased by p53 upregulated modulator of apoptosis (PUMA) and APAF-1 even though the expressions of the upstream p53-mediated apoptosis signaling proteins, BAD, BAK, BAX, NOXA, and BCL2 were suppressed. In addition, the expression of PARP-1 was increased by pamidronate whereas the expression of cleaved PARP-1 (c-PARP-1) was decreased. These results suggest pamidronate-treated RAW 264.7 cells underwent FAS/caspase 3/PARP-1-mediated apoptosis, that is, parthanatos, due to the accumulation of polymeric adenosine diphosphate ribose (poly (ADP-ribose) or PAR) caused by severe DNA damage. Actually, pamidronate-treated RAW 264.7 cells were continuously proliferative as evidenced by the up-regulations of p53/Rb/E2F and Wnt/β-catenin signaling, though they only showed a slight increase in cell numbers after 24 h of pamidronate treatment vs. non-treated controls, which suggests some cells unable to differentiate into mature macrophages may have succumbed to FAS-mediated or PARP-1-associated apoptosis.
Pamidronate reduced the expressions of the osteoclastogenesis-related proteins, RANKL and cathepsin K in RAW 264.7 cells, indicating it inhibited osteoclast differentiation, which is in-line with the reported disappearance of osteoclasts in bisphosphonate-treated animals (Kameka et al., 2014;Kawata et al., 2004;Mayahara & Sasaki, 2003) and has implications regarding the effects of pamidronate effects on osteolytic diseases such as including osteoporosis, fibrous dysplasia, Paget's disease, and Gorham's disease (Hammer et al., 2005;Kravets, 2018;Saraff et al., 2018), etc.
Pamidronate was found to widely affect the expressions of proteins in different signaling pathways in RAW 264.7 cells. Its global protein expression changes were illustrated in Fig. 8, exhibiting dynamic impacts on epigenetic modification, protein translation, RAS signaling, NFkB signaling, cellular proliferation, protection, differentiation, survival, apoptosis, inflammation, angiogenesis, and osteoclastogenesis. Highly upand down-regulated proteins for each cellular functions were summarized in Fig. 9. Pamidronate induced marked over-and under-expression of some elective proteins more than 20% compared to non-treated controls, which may play pathogenetic roles (biomarkers) for cellular differentiation, inflammation, apoptosis, angiogenesis, and osteoclastogenesis in RAW 254.7 cells.

CONCLUSIONS
Summarizing, pamidronate was found to alter the expressions of many important proteins in RAW 264.7 cells. It upregulated proliferation-related proteins associated with p53/Rb/E2F and Wnt/β-catenin signaling and inactivated epigenetic modification and protein translation. In addition, RAS (cellular growth) and NFkB (cellular stress) signalings were markedly affected by pamidronate. Pamidronate-treated cells showed that upstream of RAS signaling was stimulated by up-regulation of some growth factors, while downstream of RAS signaling was attenuated by down-regulation of ERK-1 and p-ERK-1, resulted in reduction of cMyc/MAX/MAD network expression. They also showed suppression of NFkB signaling by downregulating p38 and p-p38 and upregulating mTOR. Consequently, pamidronate affects global protein expression in RAW 264.7 cells by downregulating the expressions of immediate inflammation, cellular differentiation, survival, angiogenesis, and osteoclastogenesis-related proteins, but by upregulating PARP-1-and FAS-mediated apoptosis, protection, and proliferation-related proteins. These findings suggest pamidronate has potent anti-inflammatory, anti-angiogenesis, and anti-osteoporotic effects together with cellular stresses dysregulating RAS signaling, NFkB signaling, apoptosis, and proliferation. The present study explored the global expressions of representative essential proteins (n = 218) in pamidronate-treated RAW 264.7 cells, but some affected proteins were so dynamic and variable that they should be continuously monitored by IP-HPLC, if pamidronate treatment will be prolonged. Finally, we suggest further molecular biologic studies be undertaken on interactions between pamidronate and target proteins.