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    • The molecular target of N BPs is farnesyl

    2022-05-23

    The molecular target of N-BPs is farnesyl diphosphate synthase (FPPS), a crucial enzyme at the branch point of cholesterol and isoprenoid synthesis in the mevalonate pathway [5]. Because they bind calcium ions, N-BPs accumulate quickly in the skeleton and are internalised by osteoclasts, leading to rapid inhibition of FPPS. The subsequent lack of intracellular isoprenoid lipids prevents the post-translational prenylation of small GTPase signalling proteins such as Rap1a, leading to disruption of osteoclast function [5], [6]. The accumulation of unprenylated Rap1a can therefore be used as a biochemical indicator of the pharmacological activity of N-BPs [7], [8], [9], [10], [11]. Resistance of patients to treatment with N-BPs has been recognized as a clinical issue in cancer-associated bone disease and PDB [1], [12] where assessment of biochemical remission has been used as a measure of unresponsiveness [13]. This phenomenon is observed even in patients who have received intravenous BP therapy, thus ruling out lack of adherence to oral N-BP treatment. The molecular mechanism underlying this apparent drug resistance is unclear; upregulation of multidrug resistance protein 1, the antiapoptotic bcl-2 gene [14] and heat shock protein 27 [15] have been observed in tumour cell lines that have developed N-BP resistance, but these observations have yet to be shown to be clinically relevant. Given that FPPS is the major molecular target for N-BP drugs (reviewed in [5]), we explored the possibility, using a cellular model, that resistance to N-BPs could be acquired as a consequence of upregulation of this enzyme. Although prolonged N-BP treatment can result in increased FPPS expression in cultured Paroxetine HCl [16], [17], [18], a model system in which endogenous FPPS expression can be rapidly modulated, with the intention of altering responsiveness to N-BPs, has not been examined. Expression of endogenous FPPS, as well as other enzymes of the mevalonate pathway, is upregulated by sterol regulatory element binding proteins (SREBPs) under conditions of sterol depletion [19], [20], for example by culturing cells in the presence of lipoprotein deficient serum (LDS) [20], [21]. We therefore examined whether upregulation of endogenous FPPS, by culturing cells in LDS-containing medium, conferred resistance to the N-BP ZOL, measuring the accumulation of unprenylated Rap1a as a surrogate marker for inhibition of protein prenylation. The exact subcellular location of endogenous FPPS is not clear, with evidence for both cytosolic and peroxisomal locations [22], [23], [24], [25]. In 2007, an isoform of FPPS was identified that contains a mitochondrial targeting sequence, raising the possibility of a mitochondrial location for the enzyme [26]. We therefore examined the subcellular localisation of LDS-induced endogenous FPPS in peroxisomes, mitochondria Paroxetine HCl or the cytosol. Since HeLa cells are reported to have intact peroxisomal import pathways [27], these cells are an ideal model with which to study the subcellular localisation of FPPS.
    Materials and methods
    Results and discussion The expression of FPPS is upregulated in the absence of sterols via binding of SREBP-1 protein to the FPPS promoter [30], [31], [32]. Consistent with this, in Hela cells, as in other cell lines such as THP-1, HepG2 and Huh7 [20], [21], the absence of extracellular sterols (by culturing cells in LDS) led to an increase in the expression of endogenous FPPS (mRNA and protein). After 48h of culture in 10% [v/v] LDS, FPPS mRNA increased approximately 3.4-fold in HeLa cells compared to cells cultured in 10% [v/v] FCS, when assessed by quantitative RT-PCR (Fig. 1A). Densitometric analysis of western blots (Fig. 1B) demonstrated a 5-fold increase in 39kDa FPPS protein, when normalised to β-actin (Fig. 1C). The LDS-induced increase in FPPS was confirmed by immunofluorescence staining. FPPS was barely detectable in HeLa cells that had been cultured in 10% FCS (Fig. 2B), whereas in cells that had been cultured in 10% LDS, FPPS was clearly visible in punctate structures throughout the cytosol (Fig. 2C).