RING-box E3 Ligase Target N-terminal Lysine 55 to Regulate Turnover of Sp7 Protein

Expression plasmids and site-directed mutagenesis

Expressed proteins are either untagged or carry a small synthetic epitope at the N-terminus for easy purification and detection. The construction of mouse full-length Sp7 and deletion mutant (Δ193–428) in the pcDNA3.1 vector or in the pcDNA3.1-Flag vector is described previously (10, 12). Sp7 deletion mutant (Δ88) was generated by PCR amplification using BamHI containing forward primer 5’-at gga tcc tac cca ccc ttc cct cac tc-3’ and BGH reverse primer. The Sp7 amplicon was double digested with BamHI/XhoI and ligated with a similarly digested pcDNA3.1-Flag vector.

Full-length mouse Rbx1 and Rbx2 were amplified by PCR using a cDNA template prepared from mouse tail RNA. The coding sequence of Rbx1 was amplified using forward primer 5’-att gga tcc atg gcg gcg gcg-3’ containing an engineered BamHI and reverse primer 5’-gcc ctc cag cta atg ccc ata ctt-3’ carrying XhoI restriction sites. Rbx2 expression plasmid was constructed by a similar strategy. Rbx2 coding sequence was amplified using BamHI containing 5’-att gga tcc atg gcc gac gtg-3’ forward primer and XhoI carrying 5’-gcc ctc gag cta atg ccc ata ctt-3’ reverse primer. The PCR product was double digested with BamHI/XhoI and cloned into a similarly digested pcDNA3.1-Flag vector.

The PCR based site-directed mutagenesis approach was used to create Sp7 lysine mutants. To construct Sp7-K55A two overlapping PCR fragments were generated using wild-type Sp7 as a template. The first fragment was amplified using T7 primer located upstream of the Sp7 insert and reverse primer 5’-ccc cat ggt cgc ggg gct gaa-3’ containing the substitution mutation replacing Lysine-55 with Alanine. The second PCR fragment was generated by using 5’-ttc agc ccc cgc gac cat ggg g-3’ forward primer containing the K55A substitution and BGH reverse primer. The overlapping Sp7 fragments were combined to generate full-length Sp7 containing K55A mutation by PCR using T7 forward and BGH reverse primer. The Sp7-K55A amplicon was double digested with BamHI/XhoI and ligated into a similarly digested pcDNA3.1-Flag vector. The Sp7-K227A/K229A double mutant was generated by employing a similar strategy of overlapping PCR. The first fragment was amplified using T7 forward and 5’-cca acc gcc gcg gga gca tag ac-3’ reverse primer containing K227 and K229 lysine to alanine substitutions. The second fragment was amplified using mutation carrying 5’-gtc tat gct ccc gcg gcg gtt gg-3’ forward and BGH reverse primer. Both overlapping fragments were then combined to generate full-length Sp7 containing K227A/K229A mutations by PCR using T7 forward and BGH reverse primer. The Sp7- K227A/K229A amplicon was double digested with BamHI/XhoI and ligated into a similarly digested pcDNA3.1-Flag vector. Successful incorporation of mutation was confirmed by diagnostic digest with BstUI and sequencing. The Sp7-K55A/K227A/K229A mutant was generated by digesting in-between K55 and K227 and 3-way ligation strategy. The Sp7-K55A plasmid was digested with BamH1/Nco1 and Sp7-K227A/K229A plasmid with Nco1/XhoI to isolate the released fragments. The fragment containing K55A and fragment containing K227A/K229A mutations were then ligated into BamHI/XhoI digested pcDNA3.1-Flag vector. The positive Sp7 clone was sequenced to confirm the presence of all three mutations (K55A, K227A, K229A).

Cell culture and transient transfections

The C₃H₁₀T_1/2 and HEK293T cell lines were purchased from the ATCC. C₃H₁₀T_1/2 and HEK293T cells were selected due to the ease of transfection and a zero background of endogenous Sp7. Due to stronger adherence and spreading on the glass coverslips, C₃H₁₀T_1/2 cells were used for in-situ immunofluorescence experiments. Mesenchymal C₃H₁₀T_1/2 cells were cultured and maintained in Modified Eagle’s media (MEM) and HEK293T cells in DMEM media with high glucose. All culture media were supplemented with L-Glutamate, Ribonucleosides, 10% fetal bovine serum, and 1% penicillin-streptomycin. Cells were cultured and maintained at 37 °C in a humidified atmosphere of 5% CO₂ in air.

Transient transfections of C₃H₁₀T_1/2 cells were performed using Lipofectamine 2000 (Invitrogen, Waltham, MA) and HEK293T using PolyJet transfection reagent (SignaGen Laboratories, Rockville, MD). Cells were seeded in 6-well dishes and transfected at 80% confluency with different amounts of Sp7, Rbx1, and Rbx2 expression vectors. Transfections were carried out as per the manufacturer’s instructions. Briefly, plasmid DNA and PolyJet complexes at 1:3 ratios were formed in serum free DMEM for 15 min at room temperature and then added to the wells containing 1 ml complete media. The plasmid DNA and Lipofectamine 2000 complexes were formed at a 1:2 ratio in serum-free Opti-MEM (Gibco, Thermo Fischer, Waltham, MA) for 20 minutes. The Lipofectamine-DNA complexes were then overlayed on C₃H₁₀T_1/2 cells for 4 hours and replaced with fresh media. The cells were harvested 24 hours after transfection.

To block protein ubiquitination, transfected HEK293T cells were incubated with 20μM ubiquitination inhibitor PYR41 for 4 hours before harvest. To block proteasome transfected HEK293T cells were incubated with 50μM proteasomal inhibitor MG132 for 4 hours before harvest.

Immunoprecipitation

HEK293T cells plated in 100mm culture dishes were co-transfected with 5μg Flag-Rbx1, Flag-Rbx2, and HA-Sp7 expression plasmids. For ubiquitination, HEK293T cells were co-transfected with 5μg of Flag-Sp7, 2μg of HA-Ubiquitin in the presence or absence of 2μg of Flag-Rbx1 or Flag-Rbx2 expression plasmids. To detect ubiquitinated Sp7 protein, transfected cells were treated with 50μM MG132 for 4 hours before harvest. Transfected cells were lysed 24 later in 500μl of 0.2% NP-40 lysis buffer (150mM NaCl, 50mM Tris-HCl, 0.2% NP-40, 0.1% SDS, 0.5% SDC) supplemented with 1X cOmplete^™ protease inhibitor cocktail and brief sonication. A 50μl aliquots of cell lysates were kept aside as 10% input. The remaining cell lysates (450μl) were incubated with 45μl of magnetic beads either conjugated with mouse monoclonal anti-Flag antibody (Millipore Sigma, Burlington, MA) or 3μg mouse IgG on a shaking rotator for 2 hours at 4°C. Beads bound with immunocomplexes were collected by placing them on a magnetic rack and washed four times with ice-cold PBS supplemented with 1X cOmplete protease inhibitor. Immunoprecipitated proteins were eluted by boiling the beads in 50μl 1X SDS loading buffer (ThermoScientific, Waltham, MA). An equal volume of input, IgG, and immunoprecipitate was separated by 12% SDS-PAGE at 100V for 2 hours and transferred to a 22μm Immobilon-Fl PVDF membrane. Western blot procedure was carried out essentially as described above.

To investigate the physical interaction between endogenous Sp7 and Ring box proteins, primary osteoprogenitors were plated in 100mm dishes and cultured in osteogenic media for 18 days. The cell pellet was lysed in 500μl of 0.2% NP-40 lysis buffer. A 100μl aliquots of cell lysates were kept aside as 20% input. The remaining cell lysates (400μl) were incubated with 13μg of rabbit polyclonal Sp7 antibody or 13μg of rabbit IgG on a shaking rotator for 2 hours at 4°C. Magnetic A/G beads (10% V/V) were then added and rotated overnight at 4°C. Beads bound with immunocomplexes were collected and processed for western blotting as described above.

Biochemical Fractionation

Primary osteoprogenitors isolated from neonate mouse calvaria were seeded in 100mm plates, cultured in growth media, and harvested after two days. C₃H₁₀T_1/2 cells cultured in 100mm plates were transiently transfected with Flag-Rbx1 or Flag-Rbx2 expression vectors. Cells were pelleted 24 hours later in IXPBS containing 1X cOmplete^™ protease inhibitor cocktail. Biochemical fractionation was performed using a Nuclear-Cytosolic extraction kit (Abcam, Boston, MA). The cell pellet was resuspended in 200μl cytosolic extraction buffer (CEB) supplemented with dithiothreitol (DTT) and protease inhibitor. The cells were incubated in the CEB for 11 minutes on ice, then immediately centrifuged at 13000 rpm for 10 min at 4°C. Cytosolic fraction was transferred to a fresh, pre-chilled tube, and the nuclear pellet was resuspended in 100μl nuclear extraction buffer supplemented with DTT and protease inhibitor. Nuclei were incubated on ice for 40 minutes, with vigorous vortex every 10 minutes, followed by brief 10 seconds sonication. The nuclear fraction was centrifuged at 13000 rpm for 10 minutes at 4°C and the supernatant was transferred to the fresh pre-chilled tube. The separated fractions were resolved in 12% SDS-PAGE and blotted with respective antibodies.

Western blot analysis and antibodies

After 24 hours of transfection, cells were washed with ice-cold phosphate buffered saline (PBS) and lysed in 150μl of RIPA buffer (150mM NaCl, 50mM Tris-HCl, 2% NP-40, 0.1% Sodium Dodecyl Sulphate (SDS), 0.5% Sodium Deoxycholate (SDC)) and EDTA-free 1X cOmplete^™ protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO). Lysates were sonicated for 10 seconds, and protein was quantified by Bradford assay. Equal amounts of protein were resolved in 12% SDS-PAGE at 100V for 2 hours and transferred to 22μm Immobilon-Fl PVDF membrane (Millipore Corporation, Billerica, MA). The wet transfer was carried at 100 V for 90 minutes at 4°C. For blocking, blots were incubated at 22°C with 5% skimmed milk diluted in PBST (0.05% Tween20 in PBS) For 30 minutes. Blots were probed with primary antibodies overnight at 4°C, washed with 1XPBST four times, then probed with secondary antibodies conjugated with IRdye680 or IRdye800 (LI-COR, Lincoln, NB) for 1 hour at 22°C and finally washed four times with 1X PBST. The blots were scanned by Odyssey Imaging System (LI-COR, Lincoln, NB), and the intensity of the immunoreactive bands was quantified by LI-COR Image Studio software. Results were obtained from at least three independent experiments.

Rabbit polyclonal antibodies against Hemagglutinin (HA), Ubiquitin, Rbx1, Nucleolin, and β-actin were purchased from Cell Signaling Technologies Danvers, MA. Rabbit polyclonal antibodies against Sp7 and Rbx2 were purchased from Abcam, Waltham, Massachusetts. The mouse monoclonal antibody against Flag-epitope was purchased from Sigma, St Louis, MO, and the mouse monoclonal antibody against β-tubulin was purchased from AB clonal Woburn, MA.

In situ immunofluorescence and digital microscopy

C₃H₁₀T_½ cells were plated on sterile glass coverslips (Fisher Scientific, Pittsburgh, PA) coated with a solution containing 0.5% gelatin (Sigma Aldrich Laboratories, St. Louis, MO) in 6-well culture dishes. After 12 hours of culture, cells were transfected with Flag-Rbx1 or Flag-Rbx2 expression plasmids. A day later, transfected cells were processed for in-situ immunofluorescence. Primary calvarial osteoblasts plated on sterile gelatin coated glass coverslips for a day were subjected to in situ immunofluorescence. Cells were rinsed twice with ice-cold PBS and fixed in 3.7% formaldehyde in PBS for 10 minutes on ice. After rinsing twice with ice-cold PBS, the cells were permeabilized with 0.1% Triton X-100 (diluted in PBSA) for 20 minutes on ice. Cells were rinsed twice with PBS and then placed in PBSA (0.5% bovine serum albumin (BSA) (Fisher Bioreagents, Fair Town, NJ) in PBS.

Antibody staining was performed by incubating the cells with a 1:100 dilution of affinity purified mouse monoclonal Flag-antibody in PBSA or affinity purified rabbit polyclonal Rbx1 and Rbx2 antibodies in PBSA. Cells were incubated with antibody for 1 hour at 37°C. Coverslips were rinsed four times with ice-cold PBSA and then stained with a 1:250 dilution of Alexa 488 goat anti-mouse secondary antibody or Alexa 594 goat anti-rabbit secondary antibody (Invitrogen, Eugene, OR). Secondary antibody staining was performed for 1 hour at 37°C. Coverslips were then washed four times with ice-cold PBSA, stained with DAPI (0.5 μg added to PBSAT) for 5 minutes on ice, washed once with PBSAT (0.1% Triton X-100 diluted in PBSA), and then twice with PBS. Coverslips were then mounted with VectaShield^® Antifade Mounting Medium (Vector Laboratories, Newark, CA), air-dried for 10–15 minutes, sealed with nail polish, and stored in the dark at −20°C until imaging. Images of the cells were captured using a Keyence all-in-one fluorescence microscope.

Deconvolution and image restoration

Immunofluorescence images of primary osteoblasts or transiently transfected C₃H₁₀T½ cells were taken using ECHO revolution microscope (Discover Echo, San Diego, CA) equipped with epifluorescence filters and a Fluorescence 5MP sCMOS Mono camera. Images of cells were acquired with a 60X oil-immersed PlanApo objective with 1.42 numerical aperture at 0.25μ intervals (11–15 sections per cell) through focus (in the Z plane) with 56nm per pixel (X,Y). Theoretical Point Spread Functions (PSFs) were modeled to account for the optical characteristics of the ECHO microscope. The PSF was calculated based on the numerical aperture (1.42) of the 60X objective lens and the wavelength of the excitation light. A Gaussian distribution was used to approximate the PSF. The PSF was generated using Image Pro software to simulate the expected image distortion and to guide the deconvolution process.Images were reconstructed, and the middle section of representative cells is shown in the figures.

Rbx2-Floxed mouse model

The Rbx2^F/F model, where exon 1 of the Rbx2 gene is floxed is reported previously (36). The tamoxifen-inducible R26-CreERT2 model purchased from Jackson Lab (Strain #:008463) is reported earlier (37). Both mouse lines were maintained on the C57/BL6 background. The Rbx2^F/F mice was crossed with Rosa-CreERT mice, and their progeny intercrossed to obtain Rbx2^F/F;R26-CreERT2 mice. The genotypes of each line were confirmed by genomic DNA from tail snippets and allele specific primer pairs. The Rbx2-floxed allele was confirmed using 5′-atc agt tcc caa tgg tac ccg-3′ forward and 5′-gca aga tca tga cta ggg att g-3′ reverse primer. The presence of R26-CreERT2 allele was confirmed using forward 5′-aaa gtc gct ctg agt tgt tat-3′ and reverse 5′-cct gat cct ggc aat ttc g-3′ primers. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and conformed to relevant federal and state guidelines and regulations.

Primary osteoblast cultures, gene expression, and mineralization analysis

Primary calvarial osteoprogenitors were isolated essentially as previously published (38). Briefly, calvaria from multiple 3-day-old wild type or Rbx2^F/F; R26-CreERT2 pups were harvested and cleaned of dura and suture lines in ice-cold PBS. Frontal and parietal skull bones were cut into small fragments and digested in 5ml of fresh trypsin-collagenase solution supplemented with 1% Penicillin-streptomycin. The enzyme mixture contained 1mg/ml collagenase-P (Roche, Indianapolis, IN) and 0.25% Trypsin (Gibco, Thermo Fischer, Waltham, MA) in 1x PBS. Calvarial fragments were digested for 5 minutes at 37°C in the water bath. The supernatant from digest-1 was discarded. Fresh 7.5ml trypsin-collagenase solution was added to the calvarial tissue and incubated at 37°C for 10 minutes in a shaking water bath set at 50 rpm. Supernatant from digest-2 was strained by 100μm Swinny filter and collected in 20ml α-MEM media containing 100 μM Ribonucleosides, 2 mM L-Glutamate, 10% FBS, and 1% Penicillin-Streptomycin. The calvarial tissue was digested a third time by adding 7.5ml of the trypsin-collagenase solution, 10 minutes incubation, and straining. The media containing the digest-2 and 3 was centrifuged at 1200 rpm for 10 minutes. The primary cell pellet was resuspended in fresh α-MEM complete media and directly cultured for the osteoblast differentiation experiment. Cells were counted and 2 × 10⁵ plated in 6-well plates and 1.2 × 10⁶ cells in 100mm plates. Cells were cultured in complete α-MEM media for 3-days. For Cre activation, cultures were treated with 1μM 4-OH tamoxifen for 48 hours, changing media every 24 hours. For osteogenic induction, cultures were fed once with a complete α-MEM media containing 25μg/ml ascorbic acid and then in a complete α-MEM media containing 50μg/ml ascorbic acid and 10mM β-glycerophosphate for up to 21 days.

Cell pellets obtained from days 7, 14, and 21 of cultures were used to isolate total RNA. Cell pellets dissolved in TRIzol reagent (Thermo Fisher Scientific, Waltham, MA), were sonicated briefly and processed to extract RNA. The cDNA was prepared from 1μg of total RNA using an iScript cDNA synthesis kit (BioRad, Hercules, CA). Quantitative real-time PCR with six replicates was performed using iQ SYBR Green Supermix (BioRad) with specific primer pairs detailed in Table 1.

To assess the mineralization, osteoprogenitors obtained from Rbx2^F/F; R26-CreERT2 pups were plated in 6-well culture dishes. Tamoxifen (1μM 4-OH) treated and untreated cells were cultured in osteogenic media for 14 and 21 days. For von Kossa staining, cells were washed twice with 1XPBS and fixed with 4% formaldehyde for 10 minutes at room temperature. Cells were then washed with 1XPBS and incubated with 3% silver nitrate solution under ultraviolet light for 30 minutes. Cells were washed thrice with double distilled deionized water, and von Kossa stained plates were imaged with a digital camera.

Statistical analysis

Data were collected from at least three independent (n=3) osteoblast differentiation and transfection experiments. Independent paired t-test was used to determine the level of statistical significance. Values from gene expression and western blots from three independent experiments were used for power analysis. The G*Power 3.1 software was used with α value of 0.05 (false positive), power of 80%, and a calculated effect size around the mean. Data are presented as average with a standard error of the mean either in box and whisker plots or bar graphs. Asterisks indicate p-value *(0.05 to >0.01), **(0.01 to >0.001), ***(<0.001), **** (<0.0001).

RING-box protein E3 ligases are expressed during osteoblast differentiation and degrades Sp7 protein in osteoblasts.

The osteoblast differentiation and osteogenesis are induced by Specificity protein 7. Ubiquitination mediated protein turnover regulates the cellular levels of Sp7 protein. To investigate the expression of RING-box protein E3 ligases in skeletal tissues, RNA was harvested from 3-week-old wild-type mice. The expression of both members of the RING-box protein E3 ligase, Rbx1 and Rbx2, was noted in the femur (Fig. 1A). Interestingly, a similar level of Rbx1 and Rbx2 expression was noted in the skull. The expression of Sp7 was confirmed in the intramembranous and endochondrally derived skeletal tissues (Fig. 1A). The presence of Rbx1 and Rbx2 E3 ligases in the skull and femurs was further confirmed by western blot analysis (data not shown). To determine the expression of RING-box protein E3 ligases during osteoblast differentiation, primary calvarial osteoprogenitors were isolated from 3-day old pups. Cells cultured in osteogenic media were harvested during proliferation (day 7), matrix synthesis (day 14), and the mineralization stages (day 21).

Interestingly, both Rbx1 and Rbx2 showed a sustained expression during osteoblast differentiation (Fig. 1B). A similar pattern of expression was noted for ubiquitin, as the levels of ubiquitin mRNA remain constant during osteoblast differentiation. Consistent with the progression of osteoblast differentiation, Sp7 mRNA increased gradually, peaking at day 21 (Fig. 1B). The successful differentiation was further confirmed by significantly increased expression of Sp7 regulated osteoblast marker genes osteocalcin (Ocn) and sclerostin (Sost). Parallel plates of osteoblast differentiation time course were used to isolate whole cell proteins (Fig. 1C). Consistent with mRNA, levels of Rbx1, Rbx2 and ubiquitin proteins were generally comparable during osteoblast differentiation (Fig. 1C). Similar to the mRNA, the maximum level of Sp7 protein was noted at the mineralization stage. The densitometric signal of each protein was quantified and normalized with the respective β-tubulin signal. Ubiquitin, Rbx1, and Rbx2 protein levels showed no significant change during osteoblast differentiation. Compared to day 7, the levels of Sp7 protein were increased by 2.8-fold at day 21 of differentiation.

Having established the expression of Rbx1 and Rbx2 ligases in the skeletal tissues and osteoblasts, we examined if they regulate the turnover of Sp7 protein. Mesenchymal progenitors C₃H₁₀T_1/2 cells were co-transfected with a fixed amount of Sp7 and increasing amounts of either Rbx1 or Rbx2 expression plasmids. Interestingly, Sp7 protein was degraded in a dose dependent manner in the presence of Rbx1 (Fig. 2A). The increasing amount of Rbx1 protein was confirmed by probing blot with Flag antibody. Data quantified from three independent experiments revealed a progressively significant decrease in the amount of Sp7 protein. Nearly 85% of the Sp7 protein was degraded at the highest tested dose of Rbx1 enzyme (Fig. 2B). We also find a dose dependent decrease in the amount of Sp7 protein in the presence of Rbx2 enzyme (Fig. 2C). Data quantified from three independent experiments revealed that at the highest dose of Rbx2, ~84% of the Sp7 protein was degraded (Fig. 2D).

Targeted degradation of Sp7 protein by Rbx1 and Rbx2 E3 ligase was independently evaluated in a different cell type. Consistent with the mesenchymal progenitors, a dose dependent decrease in the Sp7 protein was noticed when Rbx1 or Rbx2 was co-expressed in the HEK293T cells (Fig. 2E). Nearly 80% of the Sp7 protein was degraded at the highest amount of Rbx1 or Rbx2. Together, these data demonstrate that both members of the RING box protein E3 ligase are expressed during osteoblast differentiation and that Rbx1 and Rbx2 target the Sp7 protein for degradation.

To rule out that RING-box E3 ligase mediated degradation of Sp7 protein is not an artifact of over expression in a non-osseous environment, we utilized primary osteoprogenitors from the Rbx2-floxed mouse model. Rbx2 gene was deleted temporally during osteoblast differentiation by tamoxifen treatment. The Cre mediated recombination efficiency was initially monitored by the in-built TdTomato reporter in the cells. Tamoxifen treatment for a day showed Cre activity in 5% of cells, which was increased to >90% of cells by day 3 (Fig. S1A). The levels of Rbx2 and Sp7 proteins were monitored at an early (day 3) and a later (day 14) stage of osteoblast maturation (Fig. 2F). By day 3, the level of Rbx2 protein was reduced by 70% in primary osteoprogenitor cultures treated with tamoxifen (Fig. 2F). Importantly, reduction in the Rbx2 protein was coupled with a 2-fold increase in the levels of Sp7 protein. Two weeks after tamoxifen treatment, Rbx2 protein was nearly absent in the primary osteoblasts. The loss of Rbx2 protein in the osteoblasts led to a 3-fold increase in the levels of Sp7 protein (Fig. 2F). Quantified signal from three independent cultures on day 14 showed a significant increase in Sp7 protein levels in the Rbx2-deficient osteoblasts (Fig. 2G). These results indicate that the endogenously expressed Rbx2 enzyme regulates the level of Sp7 protein in osteoblasts. The co-expression studies showed that Rbx1 also regulates the turnover of Sp7 protein (Fig 2A, E). Therefore, we evaluated the expression of Rbx1 protein in the Rbx2 deficient osteoblasts. Interestingly, equivalent levels of Rbx1 protein were noted in the untreated and tamoxifen treated osteoblasts at both time points (Fig. 2F). These results strongly suggest that Rbx2 E3 ligase can independently regulate the turnover of Sp7 protein in primary osteoblasts.

Rbx1 and Rbx2 enzymes are present in the cytoplasm and nucleus of osteoblast and form a molecular complex with Sp7 protein.

To define if the Rbx1 and Rbx2 mediated degradation of Sp7 protein requires the formation of a molecular complex between the two proteins, we performed co-IP experiments. Cells co-transfected with Flag-tagged Rbx1 or Rbx2 and Sp7 expression plasmids were harvested after 24 hours. Cell lysates were immunoprecipitated with either control IgG or Flag antibody-conjugated magnetic beads. We find robust pull-down of the Rbx1 protein with the Flag antibody (Fig. 3A). Importantly, the Sp7 protein was co-immunoprecipitated with the Rbx1 protein (Fig. 3A). Parallel co-IP experiment was performed with Flag-Rbx2 and Sp7 co-transfected cells. A similar co-immunoprecipitation of Sp7 and Rbx2 protein was noted (Fig. 3B). These results demonstrate that Rbx1 and Rbx2 E3 ligase form a molecular complex with the Sp7 protein. To establish the molecular interaction between endogenous Sp7 and RING-box proteins, we performed co-immunoprecipitation experiments in the primary osteoblasts. Lysates from osteoblasts were immunoprecipitated with either Sp7 antibody or control IgG. Both Rbx1 and Rbx2 E3 ligases were co-immunoprecipitated with the endogenous Sp7 protein in osteoblasts (Fig. 3C). These data confirm the physical interaction between the RING-box proteins and the Sp7 protein. Furthermore, these results establish that physical interaction observed with the overexpressed proteins is not linked with the excess availability of Sp7 and RING-box proteins in the cells.

Consistent with being a DNA binding transcription factor, the nuclear localization of the Sp7 protein is well documented. However, the cellular distribution of RING box E3 ubiquitin ligase is not known. Therefore, we performed in situ immunofluorescence experiments to examine Rbx1 and Rbx2 distribution by transiently transfecting C₃H₁₀T_1/2 cells. We observed 20–30% transfection efficiency with no cytotoxicity. Over 150 positive cells with varying expression levels of Rbx1 and Rbx2 proteins were analyzed in individual coverslips. To our surprise, Rbx1 protein was distributed in both the cell cytoplasm and the nucleus (Fig. 4, S1B). Rbx2 protein also showed a similar pattern of diffuse cytoplasmic and nuclear distribution (Fig. S1B). Interestingly, the intensity of the immunofluorescence signal in the cytoplasm and the nucleus appears equivalent, irrespective of the overall signal per cell (Fig. S1B). Cells were sectioned to acquire z-stack images to distinguish if this is a genuine nuclear distribution or represents an overlaying cytoplasmic signal. Deconvolution confirmed distinct nuclear but non-nucleolar signals of Rbx1 and Rbx2 proteins (Fig. 4A-B). Interestingly, deconvolved images revealed that in the cytoplasm, apart from the diffused and punctate signal, RING-box proteins are associated with cell membranes (Fig. 4A-B).

To quantify and independently confirm the amount of Rbx1 and Rbx2 protein in the nucleus and cytoplasm, we performed biochemical fractionation. Two plates of C₃H₁₀T_1/2 cells were transiently transfected with either Rbx1 or Rbx2. The cell pellet from one plate was used for whole cell lysates, and the second was subjected to serial biochemical fractionation to isolate cytoplasmic and nuclear proteins. Equal amounts of proteins from the whole-cell, cytoplasmic, and nuclear fractions were resolved, and blots probed with Flag antibody to detect Rbx1 and Rbx2. Consistent with the immunofluorescence data, nearly equal amount of Rbx1 protein was detected in the nuclear and cytoplasmic fractions (Fig. 4C). Rbx2 protein also showed equal distribution in the nuclear and cytoplasmic fractions (Fig. 4C). Quantification from three independent experiments revealed that 52–60% of the RING-box proteins are present in the cytoplasm and 40–48% in the nuclear fraction.

Primary osteoblasts were next used to independently confirm the cellular distribution of endogenous RING-box proteins. Both Rbx1 and Rbx2 are expressed in the osteoblasts (Fig. 1C). The endogenous Rbx1 and Rbx2 proteins were distributed throughout the nucleus and cytoplasm of the primary osteoblast in both diffused and punctate fashion (Fig. 4D-E, S1C). Deconvolved z-stack images confirmed the punctate, nuclear, and non-nucleolar distribution of both RING-box proteins in the osteoblasts (Fig. 4D-E). Interestingly, unlike the overexpressed Rbx1 and Rbx2, the endogenous proteins do not associate with the cell membranes (Fig. 4A-B). Serial biochemical fractionations were then performed to quantify the nuclear and cytoplasmic proportions of the RING-box proteins. Upon confluency, osteoblasts were harvested either for whole cell lysates or to sequentially extract the cytoplasmic and nuclear proteins. In osteoblasts, the endogenous Rbx1 and Rbx2 proteins were present in equivalent amounts in both the nuclear and cytoplasmic fractions (Fig. 4F). Quantification from three independent experiments showed 57% and 56% of Rbx1 and Rbx2 proteins were present in the cytoplasmic fraction of osteoblasts (Fig 4G). Nearly 43% of Rbx1 and 44% of the Rbx2 protein were present in the nuclear compartment (Fig 4G). Together, these data demonstrate that in addition to the cytoplasm, Rbx1 and Rbx2 enzymes are present in the nucleus and are part of the molecular complex that regulates the turnover of Sp7 protein in osteoblasts.

Rbx1 and Rbx2 E3 ligase promote ubiquitylation and degradation of Sp7 protein through the ubiquitin-proteasome pathway.

Having established the interaction and targeting of Sp7 protein by RING-box E3 ligase, we examined whether Rbx1 and Rbx2 mediated degradation involves the ubiquitin-proteasomal pathway. Cells were co-transfected with Sp7 and ubiquitin and either Rbx1 or Rbx2 expression plasmids to establish Rbx1 and Rbx2 mediated ubiquitination of Sp7. Cell lysates were used to immunoprecipitate Sp7 protein. The immunocomplexes resolved in SDS-PAGE were probed to detect ubiquitinated Sp7 protein (Fig. 5). The intensity of ubiquitinated Sp7 protein was quantified using LI-COR image studio software and presented in graphs. Surprisingly, the amount of polyubiquitinated Sp7 was reduced by 57% in the presence of Rbx1 and 50% in the presence of Rbx2 protein (Fig. 5A left panel graph). To evaluate if this decrease is linked to the degradation of ubiquitinated Sp7 protein, blots were probed with anti-Sp7 antibody (Fig. 5A left input panels). Indeed, the levels of Sp7 protein were reduced in the presence of Rbx1 and Rbx2. The proteasome is the main cellular pathway that degrades polyubiquitinated proteins. Therefore, we blocked the proteasomal pathway by treating cells with 50 μM of MG132 to quantify better the Rbx1 and Rbx2 mediated ubiquitylation of Sp7 protein. As expected, the proteasomal blockage leads to a significant accumulation of polyubiquitinated Sp7 protein (Fig. 5A right panel). The signal of polyubiquitinated Sp7 protein quantified from these blots revealed an increase of 45% in the presence of Rbx1 and 66% in the presence of Rbx2 (Fig. 5A right panel graph). Blots reprobed with Sp7 antibody further confirm the involvement of Rbx1 and Rbx2 in ubiquitination mediated turnover of Sp7 protein (Fig. 5A right input panels). Together, these data establish that RING-box E3 ligase targets Sp7 protein for ubiquitylation.

To evaluate if ubiquitination of Sp7 protein is required for Rbx1 and Rbx2 mediated degradation, we blocked the initial step of the ubiquitination pathway by chemical inhibition of ubiquitin activating enzyme Ube2. Cells co-transfected with Sp7 and increasing amounts of Rbx1 or Rbx2 were incubated with 20μM PYR-41 or DMSO. Consistent with Figure 1, the Rbx1 enzyme promoted degradation of Sp7 protein in a dose dependent fashion (Fig. 5B). However, Rbx1 mediated degradation of Sp7 protein was significantly reduced upon blockage of ubiquitination (Fig. 5B). The normalized densitometric quantification shows that Sp7 signal is reduced by 1.7-fold and 3.4-fold with 1μg and 2μg of Rbx1 respectively. Blocking the ubiquitination reduced Rbx1 mediated Sp7 degradation by 1.3-fold and 1.8-fold, respectively (compare lane 2 vs 4 and lane 3 vs 5). In the presence of 1μg and 2μg of Rbx2, levels of Sp7 protein were reduced by 1.7-fold and 2.7-fold, respectively (Fig. 5B). A similar pattern of Sp7 protein protection from Rbx2 mediated degradation was observed when protein ubiquitination was blocked (Fig. 5C). Quantification revealed a reduction in Rbx2 mediated Sp7 degradation by 1.4-fold and 1.5-fold respectively (compare lane 2 vs 4 and lane 3 vs 5). These data clearly indicate that Rbx1 and Rbx2 enzymes promote the ubiquitination dependent degradation of Sp7 protein. To investigate if ubiquitinated Sp7 protein undergoes proteasomal degradation, co-transfected cells were treated with 50μM MG132. We observed a 2.2-fold and 7.5-fold reduction in the levels of Sp7 protein in the presence of 1μg and 2μg of Rbx1, respectively (Fig. 5D). However, proteasomal inhibition reduced the Rbx1 mediated degradation of Sp7 protein by 1.2-fold and 3-fold (compare lane 2 vs 4 and lane 3 vs 5). In the presence of 1μg and 2μg of Rbx2, the levels of Sp7 protein were reduced by 2.8-fold and 18-fold, respectively (Fig. 5E). Similarly, proteasomal inhibition reduced the Rbx2 mediated degradation of Sp7 protein by 1.5-fold and 9.2-fold respectively (compare lane 2 vs 4 and lane 3 vs 5). Together, these data unequivocally establish the requirement of the ubiquitin-proteasomal pathway and the RING-box E3 ligases in regulating the turnover of Sp7 protein.

RING-box E3 ligase targets lysine 55 for ubiquitination and subsequent degradation of Sp7 protein.

After establishing the requirements of Rbx1 and Rbx2 mediated polyubiquitination and degradation of Sp7 protein, we set out to map the region of Sp7 protein targeted by these E3 ligases. Ubiquitination takes place on lysine residues in the target protein. Our in-silico analysis revealed the presence of multiple high scored putative ubiquitination sites in the Sp7 protein. Three lysines (K55, K227, K229) located in the transcriptional activation domain are evolutionary conserved among mammals (Fig. 6A). We employed a deletion mutagenesis approach and constructed an N-terminal mutant lacking the initial 88 amino-acids (Δ88) of the transcriptional activation domain but contain the 340 amino-acids in the C-terminus (89–428). The Δ88 Sp7 mutant lacks the K55 but contains the K227 and K229 (Fig. 6B). The C-terminal mutant (Δ193–428) lacks the last 236 amino acids of the Sp7 protein, including the DNA binding Zinc fingers and part of the transcriptional activation domain. The Δ193–428 Sp7 mutant expressing the initial 192 amino-acids only contains the K55 (Fig. 6B). Cells were co-transfected with either full-length, N-terminal, or C-terminal Sp7 mutants, and increasing amounts of either Rbx1 or Rbx2 expression plasmids. Consistent with earlier observations, Rbx1 degraded full-length Sp7 protein in a dose-dependent manner (Fig. 6C). Interestingly, the levels of N-terminal mutant Δ88 Sp7 protein remain unchanged despite the presence of Rbx1 enzyme (Fig. 6C). Since the Δ193–428 mutant lacks the Sp7 antibody epitope, therefore, blots were probed with Flag-antibody. We found that Δ193–428 Sp7 protein is also degraded by Rbx1 in a dose-dependent manner (Fig. 6C). These results indicate that Rbx1 targets the N-terminal region for ubiquitination and degradation of Sp7 protein. Parallel co-transfections performed with Rbx2 showed that it degrades full-length and Δ193–428 mutant Sp7 proteins in a dose-dependent manner. In contrast, the levels of Δ88 Sp7 protein remain unchanged (Fig. 6D). These results demonstrate that sequences within the transcriptional activation domain contribute to Rbx1 and Rbx2 mediated ubiquitination and degradation of Sp7 protein.

We next employed site-directed mutagenesis to identify the contributions of lysine 55, 227 and 229 in Rbx1 and Rbx2 mediated ubiquitination and degradation of Sp7 protein. To interrupt the ubiquitination of Sp7 protein, these lysine residues were substituted with alanine. Three Sp7 proteins were generated carrying either single (K55A), double (K227A/K229A), or triple (K55A/K227A/K229A) mutation in the lysine residues (Fig. 7A). Wild type and mutant Sp7 plasmids were transiently co-transfected with an increasing amount of Rbx1 or Rbx2. Both the WT and K227A/K229A Sp7 mutant proteins were progressively degraded by the Rbx1 enzyme (Fig. 7B). These results suggest that Sp7 proteins are targeted for degradation when lysine-55 is preserved. In sharp contrast, the levels of K55A and the triple mutant Sp7 proteins remained unchanged in the presence of the Rbx1 enzyme. The failure of degradation of the K55A and triple mutant Sp7 protein indicates the requirement of lysine-55 in Rbx1 mediated degradation. Consistent with these observations, both the WT and double mutant Sp7 proteins were degraded by the Rbx2 (Fig. 7B). The critical requirement of the lysine-55 was further confirmed by the lack of degradation of K55A and triple mutant by the Rbx2 enzyme (Fig. 7C). Co-IP experimnets were next performed to test if changes in the ubiquitination of lysine 55 is contributing to the impaired degradation. Cells were co-transfected with HA-ubiquitin and either WT or K55A expression plasmids. Cell lysates were immunoprecipitated with Sp7 antibody. The K55A mutant showed reduced signal with the HA-antibody in two independent Co-IP experiments (Fig. 7D). The quantified signal from the HA-probed blot was normalized with the intensity values of the respective Sp7-probed blot. Compared to the WT Sp7 protein, the K55A mutant showed a ~33–40% decrease in ubiquitination (Fig. 7E). Together, these data demonstrate that both Rbx1 and Rbx2 E3 ligases target lysine-55 to regulate the turnover of the Sp7 protein.

Rbx2 E3 ligase regulates osteoblast differentiation by targeted degradation of Sp7 protein.

The biological significance of the Sp7 protein turnover by RING-box E3 ligases was investigated using the Rbx2-floxed mouse model. The Rbx2 gene was temporally deleted in the osteoprogenitors obtained from neonatal calvarial tissues. The progression of osteoblast differentiation was monitored at two different time points by both gene expression and matrix mineralization (Fig. 8). Consistent with the gene deletion, Rbx2 mRNA and protein were both absent in the tamoxifen-treated primary osteoblasts (Fig. 8A, B). A progressive reduction in the level of early osteogenic marker alkaline phosphatase (Alp), while the progressive increase in mature osteoblast markers, Ocn and Sost, indicates the progression of osteoblast differentiation (Fig. 8A). To our surprise, Rbx2 loss in osteoblast caused no significant change in the expression of Alp on day 14. However, the expression of Alp was significantly increased in the Rbx2-deficient osteoblast by day 21 (Fig. 8A). Rbx2 deletion resulted in a significant increase in mature osteoblast markers Ocn and Sost at both day 14 and day 21. These data suggest that loss of Rbx2 E3 ligase enhances the differentiation of primary osteoblasts.

To understand the reasons for an accelerated differentiation of osteoblasts, parallel plates were processed for western blot analysis. Consistent with the earlier observations, the lack of Rbx2 was associated with the increased level of Sp7 protein in tamoxifen-treated primary osteoblasts (Fig. 8B). It is important to note that the levels of Rbx1 enzyme were unchanged in the Rbx2-deficient osteoblasts (Figs. 2F, 8B). Thus, Rbx2 E3 ligase targets Sp7 protein for ubiquitin mediated degradation independent of the Rbx1 enzyme. Changes in the expression of osteoblast marker genes prompted the examination of matrix mineralization (Fig. 8C). The von Kossa staining was performed on day 14 and day 21 of the osteoblast differentiation. A progressive increase in the mineral deposition was evident from day 14 (top) to day 21 of control osteoblast cultures (bottom). Rbx2 deletion showed a visible increase in mineral deposition at both time points (Fig. 8C). The highest von Kossa staining was observed in Rbx2-deficient osteoblast cultures at day 21. Data quantified from three independent experiments revealed that Rbx2 deletion in osteoblast led to a 40% and 20% increase in the mineral deposition at day 14 and day 21, respectively (Fig. 8D). Together, these results demonstrate that osteoblast differentiation and matrix mineralization is regulated by Rbx2 E3 ligase mediated degradation of Sp7 protein.