A highly sensitive prenylation assay reveals in vivo effects of bisphosphonate drug on the Rab prenylome of macrophages outside the skeleton

Abstract

Bisphosphonate drugs such as zoledronic acid (ZOL), used for the treatment of common bone disorders, target the skeleton and inhibit bone resorption by preventing the prenylation of small GTPases in bone-destroying osteoclasts. Increasing evidence indicates that bisphosphonates also have pleiotropic effects outside the skeleton, most likely via cells of the monocyte/macrophage lineage exposed to nanomolar circulating drug concentrations. However, no effects of such low concentrations of ZOL have been reported using existing approaches. We have optimized a highly sensitive in vitro prenylation assay utilizing recombinant geranylgeranyltransferases to enable the detection of subtle effects of ZOL on the prenylation of Rab- and Rho-family GTPases. Using this assay, we found for the first time that concentrations of ZOL as low as 10nM caused inhibition of Rab prenylation in J774 macrophages following prolonged cell culture. By combining the assay with quantitative mass spectrometry we identified an accumulation of 18 different unprenylated Rab proteins in J774 cells after nanomolar ZOL treatment, with a >7-fold increase in the unprenylated form of Rab proteins associated with the endophagosome pathway (Rab1, Rab5, Rab6, Rab7, Rab11, Rab14 and Rab21). Finally, we also detected a clear effect of subcutaneous ZOL administration in vivo on the prenylation of Rab1A, Rab5B, Rab7A and Rab14 in mouse peritoneal macrophages, confirming that systemic treatment with bisphosphonate drug can inhibit prenylation in myeloid cells in vivo outside the skeleton. These observations begin a new era in defining the precise pharmacological actions of bisphosphonate drugs on the prenylation of small GTPases in vivo.

Keywords: Rab; bisphosphonate; geranylgeranyl transferase; macrophage; prenylation.

Figure 1.

Schematic diagram of the in vitro prenylation assay. Unprenylated proteins (present in cells after bisphosphonate treatment) are prenylated in vitro in cell lysates by recombinant Rab GGTase or GGTase I, utilizing biotinylated-geranyl substrate (“B”). Hence, only unprenylated proteins become biotinylated, allowing detection on protein blots or enrichment for liquid chromatography-mass spectrometric analysis. The assay was used to analyze lysates of cultured cells and macrophages purified from ZOL-treated mice.

Figure 2.

Only the in vitro prenylation assay with Rab GGTase detects acute effects of ≤1 μM ZOL on protein prenylation. J774 macrophages were treated for 48 h with 0.5-10 μM ZOL then cell lysates were analyzed for (A) the presence of unprenylated Rap1A (red, arrowhead) and total Rap1 (green); (B) 21 kDa (eg Rho, Rac) proteins prenylated in vitro by GGTase I (arrowhead); (C) Rab proteins prenylated in vitro by Rab GGTase. The 75 kD band of endogenous biotinylated protein was used as a loading control in (B) and (C).

Figure 3.

Prolonged exposure to ZOL is required to detect effects of low nanomolar concentrations of ZOL on protein prenylation. J774 macrophages were treated with 1-500 nM ZOL for 7, 18 or 30 days. Cell lysates were then analyzed for (A) Rab proteins prenylated in vitro by Rab GGTase; (B) proteins prenylated in vitro by GGTase I (eg Rho, Rac); (C) the presence of unprenylated Rap1A (red, arrowhead) and total Rap1 (green). The loading control (75 kD band of endogenous biotinylated protein) for (A) and (B) is shown in Fig. S1. (D) 2-dimensional electrophoretic separation of in vitro prenylated Rab proteins following treatment with 500 nM ZOL for 7 days, or with 25 μM ZOL for 30 hr.

Figure 4.

ZOL treatment inhibits Rab prenylation in vivo in mouse peritoneal macrophages. Mice (3 per group) were administered either (A) a single i.p., s.c. or i.v. injection of 100 μg/kg ZOL or left untreated (cntrl), or (B) administered s.c. ZOL (n = 2 mice) or saline control (2 mice per group) once-weekly for 8 weeks. 24 hours after the final injection, cell lysates were prepared from CD11b⁺ macrophages isolated from peritoneal lavage fluid. After in vitro prenylation with Rab GGTase, samples were transferred onto blots and analyzed for the presence of biotinylated Rab proteins. The 75 kD band of endogenous biotinylated protein was used as a loading control.