Table 1.
Gene-specific primers used in this study.
Fig 1.
GRP and MGP are produced by leukocytes as γ-carboxylated proteins.
Leukocyte subsets neutrophils (Neut), monocytes (Mon), and T lymphocytes (T-Lym) were isolated from human buffy coats, and macrophages (Mac) and dendritic cells (DC) were obtained by differentiation of isolated monocytes in culture. After purity assessment by flow cytometry, these isolated leukocyte populations were used to evaluate GRP and MGP production at gene and protein levels. (A) Relative fold expression (arbitrary units) of GRP and MGP determined by qPCR in isolated leukocytes. Means are presented as replicates of 3 biological healthy donors using 18S as housekeeping gene. (B) Total protein extracts of the isolated leukocyte populations were obtained with RIPA buffer and quantified using the Micro BCA kit. Five μg, ten μg and fifteen μg of total protein extracts from T Lym, Mon and Mac, respectively, were analyzed by Western blot to detect total GRP (tGRP) and total MGP (tMGP) protein forms using the validated CTerm-GRP and tMGP antibodies, and γ-carboxylated GRP (cGRP) and MGP (cMGP) protein forms using conformation-specific antibodies. Position of relevant molecular mass markers (kDa) is indicated on the left side. (C) Qualitative gene expression analysis of the γ-carboxylated related enzymes VKOR and GGCX by RT-PCR in T lymphocytes, monocyte and neutrophils. 18S amplification was used as loading control for sample integrity.
Fig 2.
γ-carboxylated GRP and MGP are produced in THP-1 cell line.
THP-1 and THP-1 MoM differentiated with 25 ng/ml of PMA during 48h were cultured in control conditions and harvested for RNA and protein extraction. (A) Qualitative gene expression analysis of GRP, MGP, VKOR and GGCX by RT-PCR in undifferentiated THP-1 representing monocyte cells (THP-1), and in differentiated THP-1 representing macrophages (THP-1 MoM). (B) Western blot analysis of thirty μg of total RIPA protein extracts of THP-1 and THP-1 MoM cells using the conformation-specific antibodies recognizing γ-carboxylated GRP (cGRP) and MGP (cMGP). Position of relevant molecular mass markers (kDa) is indicated on the right side.
Fig 3.
GRP and MGP are up-regulated in LPS stimulated THP-1 cells.
THP-1 and differentiated THP-1 macrophage cells were stimulated with 100 ng/ml of LPS and harvested for RNA extraction at determined time points during 12h. Relative gene expression analysis of the inflammatory marker IL-1β, GRP, MGP and GGCX was determined by qPCR in monocytes (THP-1 cells) (A) and in differentiated THP-1 macrophages (B) at indicated time points. Gene expression levels are relative to the expression of control untreated cells during each time point. GAPDH was used as housekeeping gene and data is presented as means (n = 4) ± standard error of duplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P< = 0.05 (*), P< = 0.01 (**), P< = 0.001 (***) and P<0.0001 (****).
Fig 4.
GRP and MGP are up-regulated in hydroxyapatite stimulated THP-1 MoM cells.
Differentiated THP-1 macrophage cells were stimulated with 250 μg/ml of synthetic hydroxyapatite nano-crystals and harvested for RNA extraction at determined time points during 12h. Relative gene expression analysis of the inflammatory marker IL-1β, GRP and MGP was determined by qPCR at indicated time points. Gene expression levels are relative to the expression of control untreated cells during each time point. GAPDH was used as housekeeping gene and data is presented as means (n = 3) ± standard error of duplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P< = 0.05 (*), P< = 0.01 (**), and P< = 0.001 (***).
Fig 5.
GRP reduces TNFα and PGE2 production in THP-1 MoM cells stimulated with LPS.
(A) Differentiated THP-1 MoM cells were treated with 0.5 μg/ml, 0.75 μg/ml and 1.5 μg/ml of purified cGRP and ucGRP proteins for 24 h, followed by exposure to 50 ng/ml LPS for additional 24 h. Cells treated with 2 μM dexamethasone (DXM) were used as a positive anti-inflammatory control, and non-stimulated cells (C) as controls to LPS stimulation. Conditioned cell culture media were collected and used to determine TNFα accumulation by ELISA assays. Data are presented as means (n = 3) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P< = 0.05 (*), P< = 0.01 (**), and P< = 0.001 (***). (B) Differentiated THP-1 MoM cells were treated with 1.5 μg/ml of purified cGRP and ucGRP proteins for 24 h and exposed to LPS as described in (A), and PGE2 accumulation was determined in conditioned media through ELISA assays. Data are presented as means (n = 2) ± standard error of replicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P< = 0.001 (***) and P<0.0001 (****).
Fig 6.
Coating of BCPs with GRP reduces TNFα production in THP-1 MoM when compared with naked crystals.
Differentiated THP-1 MoM cells were treated with 100 μg/ml of BCP crystals or BCPs-coated with cGRP/ucGRP (PMC/cGRP; PMC/ucGRP) proteins for 24 h, and non-stimulated cells were used as control (C). Control non-stimulated cells were also treated with 1.5 μg/ml of cGRP/ucGRP proteins for comparison. Conditioned media were collected and used to determine TNFα accumulation through ELISA assays. Data are presented as means (n = 3) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P< = 0.01 (**) and P<0.0001 (****).
Fig 7.
Overexpression of GRP in THP-1 cells rescues LPS induced inflammation.
THP-1 cells were transiently transfected with GRP mRNA during 24, 48 and 72 h, and GRP overexpression was evaluated by measuring levels of GRP mRNA (A) and protein production (B). (A) GRP Normalized expression was determined by qPCR in non-transfected (C) and transfected (T) cells at the indicated time points. Data are presented as means (n = 3) ± standard error of duplicates of two independent experiments. Student’s t-test was used for comparison between C and T groups at each time point. Statistical significance was defined as P< = 0.001 (***). (B) Twenty micrograms of total protein extracts from the conditions described above were analysed by Western blot to detect tGRP and GAPDH proteins, and relative protein levels normalized to GAPDH were obtained using ImageJ software and are presented as arbitrary units. (C-G) Transfected (T) and non-transfected THP-1 (C) cells were cultured for 24 h and either maintained in control conditions (C, T) or stimulated with LPS (C-LPS, T-LPS) for additional 12 h. Normalized GRP gene expression was determined by qPCR (C), and inflammatory response was evaluated by measuring gene expression of the inflammatory markers IL-1β (D) NFkB (E) and TNFα (F) and TNFα accumulation in cell culture media by ELISA (G). (C-F) Data are presented as means (n = 6) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P< = 0.01 (**), P< = 0.001 (***) and P<0.0001 (****). ns, non-significant. (G) Data is presented as means (n = 4) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P< = 0.001 (***) and P<0.0001 (****).
Fig 8.
Overexpression of GRP in THP-1 cells rescues hydroxyapatite induced inflammation.
THP-1 cells were transiently transfected with GRP mRNA for 24 h and either maintained in control conditions (C, T) or stimulated with HA (C-HA, T-HA) for additional 48 h. Gene expression of GRP (A) and the inflammatory marker genes NFkB (B) and TNFα (C) were determined by qPCR (A), and levels of TNFα accumulation were measured by ELISA in conditioned culture media (D). (A-C) Data are presented as means (n = 6) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P< = 0.01 (**), P< = 0.001 (***) and P<0.0001 (****). ns, non-significant. (D) Data are presented as means (n = 4) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P< = 0.001 (***) and P<0.0001 (****).
Fig 9.
GRP is present in THP-1 MoM extracellular vesicles (EVs) at protein and mRNA levels.
EVs were isolated from THP-1 MoM cell conditioned media by differential ultracentrifugation at 100.000 xg and characterized by TEM (A), Western blot for the exosomal marker CD9, GAPDH and total GRP (tGRP) (B), and qualitative analysis of GRP, MGP and GAPDH mRNA (C). Scale bar in panel (a) represents 200 nm.