Abstract
Bisphosphonates (BPs) are drugs commonly used in the treatment of various disease arising or affecting bone tissue. There is a standard use in bone neoplasia and metastasis, hormonal and developmental disorders as well as for compensation of adverse effects in several medical therapies. Many in-vivo and in-vitro studies have assessed the efficacy of this drug and its function in cellular scale. In this concern, BPs are described to inhibit the resorptive function of osteoclasts and to prevent apoptosis of osteoblasts and osteocytes. They can preserve the osteocytic network, reduce fracture rate, and increase the bone mineral content, which is therapeutically used. Connexin 43 (Cx43) is a crucial molecule for basal regulation of bone homeostasis, development, and differentiation. It is described for signal transduction in many physiological and pathological stimuli and recently to be involved in BP action.
INTRODUCTION
There is a standard use of Bisphosphonate (BP) medication for the treatment of pathological changes in bone metabolism in a huge variety of diseases. Osteolytic tumors and metastasis as well as disease causing decreased bone strength and leading to resorption such as osteoporosis, glucocorticoid, radiation and chemotherapy, or congenital disorders (CitationBrumsen et al., 1997; CitationGlorieux et al., 1998; CitationLezcano et al., 2012; CitationMerrell et al., 2007; CitationYoshitani et al., 2011).
In osteoporosis and Paget's disease, the most popular first line BPs are oral Alendronate and Risedronate which are both nitrogen containing ones. Next action in patient developing gastrointestinal problems or not responding to the treatment is intravenous nitrogen-containing BP, pamidronate. It is estimated that 4 million women in US were taking BPs in 2008 (CitationSiris et al., 2011). In 2012, An estimated 14.7 million prescriptions for oral BPs were dispensed in US retail pharmacies in which 72.4% was Alendronate, 15.8% Risedronate, and 11.8% Ibandronate (CitationWysowski & Greene, 2013).
BPs are administered mostly peroral or parenteral. Local implantation of BPs seems to be an effective way in some cases. As an orally taken drug, they are prescribed as daily pills. Adverse gastrointestinal effects are the major complication of this route. Also poor gastrointestinal absorption (less than 1% or the oral dose) is its disadvantage.
It can be consumed intravenously in Paget's disease and tumor bone diseases. The interval of the latter route is weekly, biweekly, monthly, or even much longer. However, precautions must be taken while prescribing BPs through this way, having danger of renal failure (CitationEzra & Golomb, 2000).
Though being caused by differing pathological mechanisms, all mentioned diseases respond to BP medication which maintains integrity and strength in bone and reduces fracture rate. This links the mechanism of action in BPs straight to basal signaling pathways in the control of bone homeostasis. In this context, BPs were first supposed to affect osteoclastic differentiation and activity only. This model worked out for preservation of bone matrix and mineralization, but did neither explain changes in bone morphology and fracture rate that appear to be independent from mineral contents, nor dose-dependent effects in BPs (CitationPlotkin et al., 2006). Those actions as well as a general anti-apoptotic effect on osteoblasts and osteocytes are discussed to be mediated by Connexin 43 (Cx43)-derived hemichannels and linked to a phosphatase as reviewed by Bellido et al. in 2011 (CitationBellido & Plotkin, 2011). This article critically reviews the function of Cx43 in the mechanism of action in BP medication.
CONNEXIN HEMICHANNELS AND GAP JUNCTIONS IN BONE STRENGTH
Gap junction (GJ) channels are formed by two opposed hemichannels, each consisting of six molecules of Connexin (Cx). The Cx superfamily compromises of at least 21 highly conserved proteins expressed in a tissue-specific manner. Each Cx shows four membrane spanning domains, two extracellular, and one intracellular loop (CitationUnger et al., 1999). Despite these similarities, Cx subtypes mainly differ in the carboxy-terminus and channel permeability (CitationPlotkin & Bellido, 2001; CitationCao et al., 1998; CitationNiessen et al., 2000). Hemichannels of adjacent cells can align to form GJs that assemble and open transiently to allow exchange of ions and small water-soluble molecules of about less than 1 kDa (CitationEvans & Martin, 2002; CitationGoodenough & Paul, 2003).
Among these molecules, exchange of ATP can be highlighted. ATP release through connexin hemichannels has been shown under various cell conditions, including both physiology and pathology. Cx hemichannels open in response to a number of pathological stimuli, thus ATP molecules can be translocated (CitationRhett et al., 2014). Also this opening during pathologic stimuli and stress leads to disturbance of ionic homeostasis and increase in intracellular Ca2 + concentration. This results in cellular injury and death (CitationPie & Czubryt, 1995). Modification and blockade of Cxs has shown anti-inflammatory properties. CitationQiue et al. (2003) found that applying Cx43 blocker, would accelerate wound closure, reduce scarring and limit nuetrophil infiltration of the wound area, while studying incisional and excisional wounds in mice. Shintani-Ishida et al. simulated an ischemia and assessed contribution of hemichannels to ischemia–reperfusion injury in rat myocytes. They found that cell viability after ischemia was improved from 54% to 80%, by adding Gap26, a Cx43 blocker, which inhibited hemichannels opening during stress (CitationShintani-Ishida et al., 2007).
Cx43 is the major GJ protein expressed in bone tissue and mediates gap junctional intercellular communication (GJIC) among all bone cells, osteocytes, osteoblasts, and osteoclasts (CitationPlotkin et al., 2002; CitationYellowley et al., 2000). Deletion of Cx43 in early osteoblastic cell lineage exhibit defect expression of osteoblast-specific genes, low bone mass, delayed ossification, and osteoblast dysfunction, indicating that Cx43 function is required for the attainment of the full osteoblast phenotype (CitationPlotkin et al., 2008; CitationLecanda et al., 1998, Citation2000). But also mice lacking type XII collagen showed a decrease of functional coupling, Cx43 expression and osteoblastic differentiation giving a hint to interactions between the osteocyte network and the surrounding matrix (CitationIzu et al., 2011).
Bone strength is not only caused by mineralization. About 40% is a result of other factors like bone size and architecture of cortical and cancellous compartments of the bone matrix, the presence of micro damage and osteocyte apoptosis (CitationWeinstein, 2000; CitationFelsenberg & Boonen, 2005; CitationVashishth et al., 2000). A functional osteocyte network is provided by GJIC in osteocytes among the mineralized bone matrix. Osteocyte death and disruption of this network can compromise this mechanism leading to an accumulation of micro damage increasing bone fragility (CitationPlotkin et al., 2006; CitationPlotkin & Bellido, 2001; CitationVashishth et al., 2000). This appears important as the prevalence of osteocyte apoptosis is augmented in conditions associated with increased risk of bone fractures such as glucocorticoid-induced osteoporosis and sex steroid deficiency, which is both successfully treated with BPs (CitationPlotkin et al., 2002; CitationWeinstein, 2000; CitationWeinstein et al., 1998). Cx43 appears to be responsible for synchronized cell actions at tissue level, as observed to be indispensable in bone development, remodeling, and recovery (CitationIzu et al., 2011; CitationWatkins et al., 2012; CitationChaible et al., 2011; CitationAmano et al., 2012). In this context, Cx43-derived hemichannels and GJIC appears to be necessary for basal survival signaling mediated by Src and Erk kinase as well as for signal transduction in physiological stimuli and noxae and for the regulation of osteoclastic differentiation and function (CitationPlotkin & Bellido, 2001; CitationPlotkin et al., 2002; CitationKylmäoja et al., 2013).
BPs
BPs are stable analogues of natural inorganic pyrophosphates with a small molecular size (< 300 Da), sharing a pyrophosphate-derived P-C-P backbone structure which binds hydroxyapatite and is integrated to the calcified bone matrix. Two side chains attached to the carbon atom are responsible for the differing effects observed in BPs (CitationRussell et al., 2008). According to the nitrogen content of those side chains, BPs are classified as aminobisphosphonates or nitrogen-containing BPs (NCBPs), and non-aminobisphosphonates.
Both BP subtypes affect osteoclasts. In this concern they cause apoptosis, decreased resorption activity, recruitment, and progenitor development in osteoclasts and decrease bone remodeling (CitationPlotkin et al., 2006, Citation2008). NCBPs such as Pamidronate, Alendronate, Residronate, and Zoledronate inhibit farensyl pyrophosphate synthase in the mevalonate pathway (CitationRogers, 2004; Citationvan Beek et al., 1999). Pyrophosphate resembling BPs such as Clodronate and Etidronate are metabolized into toxic ATP-like analogues (CitationLehenkari et al., 2002; CitationPlotkin et al., 2006).
In osteoblasts and osteocytes, there is a dose- dependent response in amino- and non-aminobisphosphonate medication. There is a maximal anti-apoptotic effect on osteoblasts and osteocytes observed in a concentration between 10−6 and 10−8 M. Maximal pro-apoptotic effect of this drug has shown to be in a concentration of 10−4–10−5 M (6). Higher concentrations of BPs ≥ 10−5 M have inhibitory effects on differentiation and proliferation of osteoblastic cells (CitationBellido & Plotkin, 2011). Low concentrations of BPs have been reported to increase viability in osteoblastic cell lines and human bone marrow stromal cells and seem to have a direct impact on bone strength and resistance to fragility (CitationIm et al., 2004; CitationPlotkin & Bellido, 2001; Citationvon Knoch et al., 2005; CitationXiong et al., 2009; CitationYoshitani et al., 2011).
THE ROLE OF CX43 IN BP MEDICATION (brief in )
Alendronate mediates opening of Cx43 hemichannels
Lucifer yellow (LY), a fluorescent dye which is impermeable to the cell membrane, shows a Cx43 hemichannel-mediated dye uptake. Addition of Alendronate or EGTA, an established maneuver that opens CX hemichannel via removal of extracellular Ca2+, resulted in increased LY uptake in both, adherent cells and cells maintained in suspension (CitationLi et al., 1996; CitationPfahnl & Dahl, 1999). Dye uptake was significantly decreased using agents like AGA that disassemble Cx hemichannels. These results indicate the presence of functional Cx43 hemichannels in osteocytic cells that open after Alendronate exposure without affecting GJIC (CitationPlotkin & Bellido, 2001; CitationPlotkin et al., 2002). This opening may result from direct or indirect interaction of BPs with Cx43 pore-forming region, which leads to alteration of the tertiary and/or quaternary structures of Cx43, but could also be a result of local decrease of the extracellular calcium level (CitationLezcano et al., 2012).
Table 1. Brief synopsis of articles reviewed fielded on relevant subject.
BP uptake and binding
In endocytic cell types such as macrophages, CD14 bone marrow monocytes and osteoclasts, BPs are internalized by fluid phase endocytosis (CitationRoelofs et al., 2010). In calcified tissues, most of BP uptake in osteoclasts is suggested to be linked to resorption and endocytosis of bone matrix which BPs are bound to. CitationLezcano et al. (2012) studied the internalization of BPs by non-resorptive bone cells like osteoblastic cells. Alendronate fluorescently labeled with Alexa Flour 488 (AF-ALN) and cells were incubated in the presence of AF-ALN. Alendronate was distributed throughout the cytosol, mostly to the peri-nuclear region. Cx43 deficient HeLa cells also showed ALN uptake which indicates that Cx43 is dispensable for osteoblastic BPs internalization.
Boland et al. suggested BPs to bind sites in the cell surface modulating calcium influx via voltage-depending calcium channels by inhibition of protein phosphatases. This was observed in osteoblasts at olpadronate and p-nitrophenylphosphate (p-npp) stimulation, which is a phosphatase substrate (CitationBoland et al., 2003). Lezcano et al. investigated BP binding to osteoblastic cells by competitive binding assays with tritiated Alendronate, again showing binding sites on the cell surface (CitationLezcano et al., 2012; CitationBoland et al., 2003). In competitive binding assays using Cx43 deficient HeLa and osteoblastic cells treated with agents that disassembles Cx hemichannels, the specific binding of tritiated Alendronate to osteoblastic cells appeared to happen independently from Cx43 (CitationLezcano et al., 2012). In silencing the expression of Cx43 using s-RNA (Short Hairpin RNA Lentiviral Particles), no significant differences of BP binding to osteocytic cells was found which again indicates that there is no Cx43 involvement in BP binding.
Lezcano et al. showed in continuation to Boland et al., that the anti-apoptotic effect of BPs in HeLa and MLO-Y4 osteocyte-like cells was again reversed in the presence of p-npp; in this concern, the phosphatase is supposed to interact with the regulatory c-terminal tail of Cx43 (CitationLezcano et al., 2012). Gurley et al. suggested a molecular candidate for a pyrophosphate transporter, ANKH, as BPs uptake might occur via a ppi transporter as they resemble to the naturally occurring pyrophosphate (ppi; CitationGurley et al., 2006). There is growing evidence that the ANKH transmembrane protein may be involved in the regulation of calcium precipitation in several tissues, including joints, bones, and the kidneys (CitationCarr et al., 2008).
Induction of survival signals
The anti-apoptotic effect of BPs on osteoblastic and osteocytic cells depends on Cx43 expression, presence of functional Cx43, and channel-opening (CitationBellido & Plotkin, 2011; CitationPlotkin & Bellido, 2001; CitationPlotkin et al., 2005, Citation2008). Both, amino and non-amino BPs show an indirect anti-apoptotic effect on osteoblasts and osteocytes which is mediated by Cx43-derived activation of Src tyrosine kinase and ERKs (extracellular signal regulated kinases; CitationBellido & Plotkin, 2011; CitationPlotkin & Bellido, 2001; CitationPlotkin et al., 2005, Citation2008). According to this, Plotkin et al. observed that BPs could not prevent etoposide- or glucocorticoid-induced apoptosis in Cx43deficient HeLa and osteoblastic cells while they abolished apoptosis in HeLa cells transfected with Cx43 (CitationPlotkin et al., 2006, Citation2008). The anti-apoptotic effect of BPs in MLO-Y4 osteocytic cells was abrogated using AGA to disassembles Cx channels or via induction of GJ closure using carbenoxolone and oleamide (CitationPlotkin et al., 2002). In absence of GJIC in MLO-Y4 cells due to low cell density or maintaining cells in suspension, Alendronate prevented dexamethasone-induced apoptosis (CitationPlotkin & Bellido, 2001; CitationPlotkin et al., 2002). Therefore, CitationPlotkin et al. (2008) suggested that the anti-apoptotic effect of BPs is mediated by hemichannel action only and does not require GJIC.
Cx43 appears to be the only Cx affected by BP- mediated survival signal as observed in HeLa cells transfected with an expression vector containing Cx43, Cx26, Cx31, Cx32, Cx37, Cx40, or Cx45. In this concern, Cx45 and Cx43 co-expressions repressed the anti-apoptotic effect which could be part of the permeability decreasing interaction between Cx45, Cx43, and zonula-occludens-protein-1 (CitationLaing et al., 2001). However, these results indicate that Cx43 is the only channel protein required for transduction of survival signals (CitationPlotkin & Bellido, 2001; CitationPlotkin et al., 2002). Presence and integrity of both, pore-forming parts and C-terminal tail of Cx43 is necessary for anti-apoptotic effects in BPs as observed in truncated mutants of Cx43 which lacked pore-forming region or C-terminal (CitationBellido & Plotkin, 2011; CitationPlotkin & Bellido, 2001; CitationPlotkin et al., 2002; CitationZhou et al., 1999). Bellido et al. compared anti-apotptotic effects in several BP analogues to those found in osteoclasts. The analog IG9402, which has no effect in osteoclasts at all, not only prevented osteoblast and osteocyte apoptosis, but also preserved the decrease in bone mineral density, bone volume, osteoblast number, and bone formation rate induced by glucocorticoids (CitationBellido & Plotkin, 2011). Another investigation observed inhibition of etoposide-induced apoptosis in osteocytic cells in all 16 BP analogues while 10 of them lead to induction of osteoclast apoptosis (CitationPlotkin et al., 2006). This model tells that the two distinct effects observed in BPs, the effect on osteoclastic and on the osteocytic lineage, appear to be independent.
Effects of BPs on proliferation and differentiation
BPs stimulate DNA synthesis in osteocytes and osteoblasts independently from Cx43. CitationLezcano et al. (2012) supported this notion using ROS 17/2.8 cells and [3H]-thymidine incorporation assays. They used phosphatase inhibitors (NaF, Na3VO4) as positive controls of cellular proliferation (CitationMorelli et al., 2011). To evaluate Cx43 dependency, a Cx43-deficient HeLa cell control was used. Treatment of HeLa cells with ALN resulted in a dose-dependent increase of cell proliferation, which appeared to be independent from Cx43.
Low concentrations of BPs increase bone resorption, cell proliferation, and cell motility in osteoblastic sarcoma cell line due to Cx43-mediated effects on cell viability. Measuring Cathepsin K activity, there was no significant difference using BPs. This gives a hint to proliferative effects and increase of cell motility to be responsible for the increased bone resorption, as Cx43 mRNA expression was markedly increased after treatment. Application of oleamide, which is a potent Cx43 inhibitor, led to decreased proliferation of HOS-cells.
Prevention of bone loss by BPs
Decreased osteoblast apoptosis significantly increases the number of osteoblasts present at bone formation sites (CitationBellido & Plotkin, 2011). Mice over-expressing anti-apoptotic BCL-2 protein exhibited increased bone volume/total volume (BV/TV) in bone tissue comparing to wild-type mice and appeared to be protected from bone loss caused by ageing (CitationPantschenko et al., 2005). Though, mice lacking Caspase-3, a molecule crucial for apoptosis, also exhibited decreased BV/TV which points to further factors leading to preservation of bone tissue (CitationMiura et al., 2004). In vitro, apoptosis of osteoblasts in bone-like tissue appears to be an obligate part of the regular tissue development (CitationLynch et al., 1998). Referring to previous studies, it seems there is an inverse relationship between osteoblast survival and mineralization capability; performing mechanical tests, bone strength was assessed and it was proved that administration of IG9402 prevents bone fragility without significantly affecting the rate of bone remodeling (CitationBellido & Plotkin, 2011). However, the administration of BPs induces an increase rather than decrease in bone mineralization (CitationGourion-Arsiquaud et al., 2010). Delayed osteoblast apoptosis might result in an increased work time of osteoblast, leading to gradual increase in trabecular thickness observed in animals or patients upon long-term treatment with BPs (CitationFelsenberg & Boonen, 2005). Moreover, prevention of osteoblast and osteocyte apoptosis by BPs, even in case of bone mass loss, is shown to sufficiently maintain bone strength (CitationO’Brien et al., 2004).
CitationPlotkin et al. (2008) observed that Alendronate increases bone mineral content and prevents prednisolone- induced bone loss independent from Cx43 expression and without significant differences in bone acquisition in vivo. These findings indicate that inhibition of osteoclastic resorption is the predominant effect of Alendronate in the early phase of bone loss induced by glucocorticoids. CitationWatkins et al. (2012) observed a comparable gain of trabecular bone mass and cortical thickness in Alendronate and Risedronate treatment of ovariectomized and Cx43-deficient ovariectomized mice. Long-term treatment with BPs increases wall thickness, an index of focally increased osteoblast numbers or activity resulting in a more complete refilling of resorption cavities (CitationBalena et al., 1993; CitationChavassieux et al., 1997; CitationHorie et al., 2003; CitationStorm et al., 1993). BPs also increased bone volume and trabecular thickness in dogs, rats, minipigs, and humans (CitationBorah et al., 2002; CitationHu et al., 2002; CitationMashiba et al., 2001; CitationJeong et al., 2013).
CONCLUSION
The major effect in BPs concerns the bone matrix and osteoclasts, as BPs are rapidly integrated to the calcified bone matrix which is following endocited by osteoclasts affecting function, differentiation and recruitment. Secondary effects take place in non-resorbing bone cells and can be summarized to Cx43 derived and independent actions. In this concern, BP uptake and a basal proliferative action appears to be independent from Cx43 in all non-resorptive bone cells, but BP uptake seems to affect opening states of hemichannels via manipulation of Cx-phosphorylation which leads to anabolic and anti-apoptotic signaling. As Alendronate and Risedronate show similar effects in Cx43-deficient rodents as observed by Watkins et al. and Plotkin et al., effects on Cx43 seem to be circumstantial (6, 24). BPs bind and cause changes in the bone matrix, which could also modulate Cx43 expression, hemichannel function, and GJIC. It is uncertain whether BPs are able to modulate hemichannel function only, as hemichannels assemble and dissemble transiently to GJs and also changes in Cx43 expression affects both.
BP action in Cx43 appears to be hardly investigated yet. Studies mainly appear to be addressed to the aminobisphosphonates, Alendronate and Risedronate, which are prescribed most often. However, other nitrogen-containing BPs and non-nitrogen BPs should be examined. One workgroup holds most of the data and also the only review published in 2011.
According to this, there is not much evidence on BP action in Cx43-derived structures.
Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES
- Amano K, Ishiguchi M, Aikawa T, Kimata M, Kishi N, Fujimaki T, Murakami A, Kogo M (2012). Cleft lip in oculodentodigital dysplasia suggests novel roles for connexin43. J Dent Res. 91: S38–S44.
- Balena R, Toolan B, Shea M, Markatos A, Myers E, Lee S, Opas E, Seedor J, Klein H, Frankenfield D (1993). The effects of 2-year treatment with the aminobisphosphonate alendronate on bone metabolism, bone histomorphometry, and bone strength in ovariectomized nonhuman primates. J Clin Invest. 92: 2577.
- Bellido T, Plotkin LI (2011). Novel actions of bisphosphonates in bone: preservation of osteoblast and osteocyte viability. Bone. 49: 50–55.
- Boland R, Santillan G., Katz S, Morelli S, Stockman G, Roldan E (2003). Modulation of [Ca2+] in osteoblastic cells by olpadronate and amino-olpadronate. Protein phosphatases as possible target of bisphosphonates. Biocell. 27: 132.
- Borah B, Dufresne TE, Chmielewski PA, Gross GJ, Prenger MC, Phipps RJ (2002). Risedronate preserves trabecular architecture and increases bone strength in vertebra of ovariectomized minipigs as measured by three-dimensional microcomputed tomography. J Bone Miner Res. 17: 1139–1147.
- Brumsen C, Hamdy NA, Papapoulos SE (1997). Long-term effects of bisphosphonates on the growing skeleton: studies of young patients with severe osteoporosis. Medicine. 76: 266–283.
- Cao F, Eckert R, Elfgang C, Nitsche JM, Snyder SA, Hulser DF, Willecke K, Nicholson BJ (1998). A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J Cell Sci. 111: 31–43.
- Carr G, Sayer J, Simmons N (2008). Expression and localisation of the pyrophosphate transporter, ANK, in murine kidney cells. Cell Physiol Biochem. 20: 507–516.
- Chaible LM, Sanches DS, Cogliati B, Mennecier G, Dagli MLZ (2011). Delayed osteoblastic differentiation and bone development in Cx43 knockout mice. Toxicol Pathol. 39: 1046–1055.
- Chavassieux PM, Arlot ME, Reda C, Wei L, Yates AJ, Meunier PJ (1997). Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis. J Clin Invest. 100: 1475.
- Evans WH, Martin PE (2002). Gap junctions: structure and function (Review). Mol Membr Biol. 19: 121–136.
- Ezra A, Golomb G (2000). Administration routes and delivery systems of bisphosphonates for the treatment of bone resorption. Adv Drug Deliv Rev. 42: 175–195.
- Felsenberg D, Boonen S (2005). The bone quality framework: determinants of bone strength and their interrelationships, and implications for osteoporosis management. Clin Ther. 27: 1–11.
- Glorieux FH, Bishop NJ, Plotkin H, Chabot G, Lanoue G, Travers R (1998). Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med. 339: 947–952.
- Goodenough DA, Paul DL (2003). Beyond the gap: functions of unpaired connexon channels. Nat Rev Mol Cell Biol. 4: 285–295.
- Gourion-Arsiquaud S, Allen MR, Burr DB, Vashishth D, Tang SY, Boskey AL (2010). Bisphosphonate treatment modifies canine bone mineral and matrix properties and their heterogeneity. Bone. 46: 666–672.
- Gurley KA, Reimer RJ, Kingsley DM (2006). Biochemical and genetic analysis of ANK in arthritis and bone disease. Am J Hum Genet. 79: 1017–1029.
- Horie D, Takahashi M, Aoki K, Ohya K (2003). Clodronate stimulates bone formation as well as inhibits bone resorption and increases bone mineral density in rats fed a low-calcium diet. J Med Dental Sci. 50: 121–132.
- Hu J, Ding M, Søballe K, Bechtold J, Danielsen C, Day J, Hvid I (2002). Effects of short-term alendronate treatment on the three-dimensional microstructural, physical, and mechanical properties of dog trabecular bone. Bone. 31: 591–597.
- Im G-I, Qureshi SA, Kenney J, Rubash HE, Shanbhag AS (2004). Osteoblast proliferation and maturation by bisphosphonates. Biomaterials. 25: 4105–4115.
- Izu Y, Sun M, Zwolanek D, Veit G, Williams V, Cha B, Jepsen KJ, Koch M, Birk DE (2011). Type XII collagen regulates osteoblast polarity and communication during bone formation. J Cell Biol. 193: 1115–1130.
- Jeong HM, Jin Y-H, Choi Y-H, Chung JO, Cho DH, Chung MY, Civitelli R, Chung DJ, Lee KY (2013). Risedronate increases osteoblastic differentiation and function through connexin43. Biochem Biophys Res Commun. 432: 152–156.
- Kylmäoja E, Kokkonen H, Kauppinen K, Hussar P, Sato T, Haugan K, Larsen BD, Tuukkanen J (2013). Osteoclastogenesis is influenced by modulation of gap junctional communication with antiarrhythmic peptides. Calcif Tissue Int. 92: 270–281.
- Laing JG, Manley-Markowski RN, Koval M, Civitelli R, Steinberg TH (2001). Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells. J Biol Chem. 276: 23051–23055.
- Lecanda F, Towler DA, Ziambaras K, Cheng S-L, Koval M, Steinberg TH, Civitelli R (1998). Gap junctional communication modulates gene expression in osteoblastic cells. Mol Biol Cell. 9: 2249–2258.
- Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R (2000). Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol. 151: 931–944.
- Lehenkari PP, Kellinsalmi M, NäPänkangas JP, Ylitalo KV, Mönkkönen J, Rogers MJ, Azhayev A, VääNänen HK, Hassinen IE (2002). Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite. Mol Pharmacol. 61: 1255–1262.
- Lezcano V, Bellido T, Plotkin L, Boland R, Morelli S (2012). Role of connexin 43 in the mechanism of action of alendronate: Dissociation of anti-apoptotic and proliferative signaling pathways. Arch Biochem Biophys. 518: 95–102.
- Li H, Liu T-F, Lazrak A, Peracchia C, Goldberg GS, Lampe PD, Johnson RG (1996). Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells. J Cell Biol. 134: 1019–1030.
- Lynch MP, Capparelli C, Stein JL, Stein GS, Lian JB (1998). Apoptosis during bone-like tissue development in vitro. J Cell Biochem. 68: 31–49.
- Mashiba T, Turner C, Hirano T, Forwood M, Johnston C, Burr D (2001). Effects of suppressed bone turnover by bisphosphonates on microdamage accumulation and biomechanical properties in clinically relevant skeletal sites in beagles. Bone. 28: 524–531.
- Merrell MA, Wakchoure S, Ilvesaro JM, Zinn K, Gehrs B, Lehenkari PP, Harris KW, Selander KS (2007). Differential effects of Ca2 + on bisphosphonate-induced growth inhibition in breast cancer and mesothelioma cells. Eur J Pharmacol. 559: 21–31.
- Miura M, Chen X-D, Allen MR, Bi Y, Gronthos S, Seo B-M, Lakhani S, Flavell RA, Feng X-H, Robey PG (2004). A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells. J Clin Invest. 114: 1704–1713.
- Morelli S, Bilbao PS, Katz S, Lezcano V, RoldáN E, Boland R, Santillan G (2011). Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation. Arch Biochem Biophys. 507: 248–253.
- Niessen H, Harz H, Bedner P, Kramer K, Willecke K (2000). Selective permeability of different connexin channels to the second messenger inositol 1, 4, 5-trisphosphate. J Cell Sci. 113: 1365–1372.
- O’Brien CA, Jia D, Plotkin LI, Bellido T, Powers CC, Stewart SA, Manolagas SC, Weinstein RS (2004). Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology. 145: 1835–1841.
- Pantschenko AG, Zhang W, Nahounou M, Mccarthy MB, Stover ML, Lichtler AC, Clark SH, Gronowicz GA (2005). Effect of osteoblast-targeted expression of Bcl-2 in bone: differential response in male and female mice. J Bone Miner Res. 20: 1414–1429.
- Pfahnl A, Dahl G (1999). Gating of cx46 gap junction hemichannels by calcium and voltage. Pflügers Arch. 437: 345–353.
- Pie GN, Czubryt MP (1995). The contribution of ionic contribution of ionic imbalance to ischemia/reperfusion-induced injury. J Mol Cell Cardiol. 27: 53–63.
- Plotkin L, Bellido T (2001). Bisphosphonate-induced, hemichannel-mediated, anti-apoptosis through the Src/ERK pathway: a gap junction-independent action of connexin43. Cell Commun Adhes. 8: 377–382.
- Plotkin LI, Aguirre JI, Kousteni S, Manolagas SC, Bellido T (2005). Bisphosphonates and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms downstream of extracellular signal-regulated kinase activation. J Biol Chem. 280: 7317–7325.
- Plotkin LI, Lezcano V, Thostenson J, Weinstein RS, Manolagas SC, Bellido T (2008). Connexin 43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo. J Bone Miner Res. 23: 1712–1721.
- Plotkin LI, Manolagas SC, Bellido T (2002). Transduction of cell survival signals by connexin-43 hemichannels. J Biol Chem. 277: 8648–8657.
- Plotkin LI, Manolagas SC, Bellido T (2006). Dissociation of the pro-apoptotic effects of bisphosphonates on osteoclasts from their anti-apoptotic effects on osteoblasts/osteocytes with novel analogs. Bone. 39: 443–452.
- Qiu C, Coutinho P, Frank S, Franke S, Law L-Y, Martin P, Green CR, Becker DL (2003). Targeting connexin43 expression accelerates the rate of wound repair. Curr Biol. 13: 1697–1703.
- Rhett JM, Fann SA, Yost MJ (2014). Purinergic signaling in early inflammatory events of the foreign body response: modulating extracellular ATP as an enabling technology for engineered implants and tissues. Tissue Eng Part B Rev.
- Roelofs AJ, Coxon FP, Ebetino FH, Lundy MW, Henneman ZJ, Nancollas GH, Sun S, Blazewska KM, Bala JLF, Kashemirov BA (2010). Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in vivo. J Bone Miner Res. 25: 606–616.
- Rogers M (2004). From molds and macrophages to mevalonate: a decade of progress in understanding the molecular mode of action of bisphosphonates. Calcif Tissue Int. 75: 451–461.
- Russell R, Watts N, Ebetino F, Rogers M (2008). Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos Int. 19: 733–759.
- Shintani-Ishida K, Uemura K, Yoshida K-I (2007). Hemichannels in cardiomyocytes open transiently during ischemia and contribute to reperfusion injury following brief ischemia. Am J Physiol Heart Circ Physiol. 293: H1714–H1720.
- Siris ES, Pasquale MK, Wang Y, Watts NB (2011). Estimating bisphosphonate use and fracture reduction among US women aged 45 years and older, 2001–2008. J Bone Miner Res. 26: 3–11.
- Storm T, Steiniche T, Thamsborg G, Melsen F (1993). Changes in bone histomorphometry after long-term treatment with intermittent, cyclic etidronate for postmenopausal osteoporosis. J Bone Miner Res. 8: 199–208.
- Unger VM, Kumar NM, Gilula NB, Yeager M (1999). Three-dimensional structure of a recombinant gap junction membrane channel. Science. 283: 1176–1180.
- van Beek E., Pieterman E, Cohen L, Löwik C, Papapoulos S (1999). Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem Biophys Res Commun. 264: 108–111.
- Vashishth D, Verborgt O, Divine G, Schaffler M, Fyhrie D (2000). Decline in osteocyte lacunar density in human cortical bone is associated with accumulation of microcracks with age. Bone. 26: 375–380.
- von Knoch F., Jaquiery C, Kowalsky M, Schaeren S, Alabre C, Martin I, Rubash HE, Shanbhag AS (2005). Effects of bisphosphonates on proliferation and osteoblast differentiation of human bone marrow stromal cells. Biomaterials. 26: 6941–9.
- Watkins MP, Norris JY, Grimston SK, Zhang X, Phipps RJ, Ebetino FH, Civitelli R (2012). Bisphosphonates improve trabecular bone mass and normalize cortical thickness in ovariectomized, osteoblast connexin43 deficient mice. Bone. 51: 787–794.
- Weinstein RS (2000). True strength. J Bone Miner Res. 15: 621–625.
- Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC (1998). Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest. 102: 274.
- Wysowski DK, Greene P (2013). Trends in osteoporosis treatment with oral and intravenous bisphosphonates in the United States, 2002–2012. Bone. 57: 423–428.
- Xiong Y, Yang H, Feng J, Shi Z, Wu L (2009). Effects of alendronate on the proliferation and osteogenic differentiation of MG-63 cells. J Int Med Res. 37: 407–416.
- Yellowley CE, Li Z, Zhou Z, Jacobs CR, Donahue HJ (2000). Functional gap junctions between osteocytic and osteoblastic cells. J Bone Miner Res. 15: 209–217.
- Yoshitani K, Kido A, Honoki K, Akahane M, Fujii H, Tanaka Y (2011). Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation. Pathol Res Pract. 207: 417–422.
- Zhou L, Kasperek EM, Nicholson BJ (1999). Dissection of the molecular basis of pp60v-src induced gating of connexin 43 gap junction channels. J Cell Biol. 144: 1033–1045.