The role of vesicle trafficking genes in osteoblast differentiation and function

Using Col2.3GFP transgenic mice expressing GFP in maturing osteoblasts, we isolated Col2.3GFP+ enriched osteoblasts from 3 sources. We performed RNA-sequencing, identified 593 overlapping genes and confirmed these genes are highly enriched in osteoblast differentiation and bone mineralization annotation categories. The top 3 annotations are all associated with endoplasmic reticulum and Golgi vesicle transport. We selected 22 trafficking genes that have not been well characterized in bone for functional validation in MC3T3-E1 pre-osteoblasts. Transient siRNA knockdown of trafficking genes including Sec24d, Gosr2, Rab2a, Stx5a, Bet1, Preb, Arf4, Ramp1, Cog6 and Pacs1 significantly increased mineralized nodule formation and expression of osteoblast markers. Increased mineralized nodule formation was suppressed by concurrent knockdown of P4ha1 and/or P4ha2, encoding collagen prolyl 4-hydroxylase isoenzymes. MC3T3-E1 pre-osteoblasts with knockdown of Cog6, Gosr2, Pacs1 or Arf4 formed more and larger ectopic mineralized bone nodules in vivo, which was attenuated by concurrent knockdown P4ha2. Permanent knockdown of Cog6 and Pacs1 by CRISPR/Cas9 gene editing in MC3T3-E1 pre-osteoblasts recapitulated increased mineralized nodule formation and osteoblast differentiation. In summary, we have identified several vesicle trafficking genes with roles in osteoblast function. Our findings provide potential targets for regulating bone formation.


Expression of prolyl 4-hydroxylase subunits increases with vesicle trafficking gene knockdown
Since these trafficking genes encode ER-and/or Golgi-associated proteins that participate in vesicular transport, we examined whether knockdown of these trafficking genes affected ER homeostasis.We found that expression of at least one of the ER stress sensors (Xbps1, GRP78/Bip and Chop) was significantly increased with knockdown of Sec24d, Aplp1, Gosr2, Rab2a, Stx5a, Bet1, Preb, Arf4, Ramp1, Golga4, Cog6 or Pacs1 (Fig. 4).Disruption of ER homeostasis may lead to accumulation of misfolded proteins and the unfolded protein response 12,13 .

Vesicle trafficking gene knockdown increases mineralized nodule formation in vivo
To examine the effects of vesicle trafficking gene knockdown on mineralized nodule formation in vivo, we knocked down Gosr2, Arf4, Cog6 and Pacs1 (selected for the most consistency of in vitro phenotypes) with and without concurrent knockdown of P4ha2 in MC3T3-E1 pre-osteoblasts, which were mixed with Matrigel and implanted subcutaneously into immunodeficient mice.Mice were euthanized at 8 weeks and implants were analyzed by X-ray and micro-CT.The number of mineralized nodules formed was significantly higher with knockdown of Gosr2, Arf4, Cog6 or Pacs1 compared to control (Fig. 7A, B).While concurrent knockdown of P4ha2 did not significantly reduce the numbers of mineralized nodules compared to knockdown of Gosr2, Arf4, Cog6 or Pacs1 alone, mineralized lump volume and micro-CT total intensity were significantly decreased (Fig. 7C).We confirmed mineralization by histological analysis of nodules stained with Alizarin Red S and Von Kossa (Fig. 7D).Collectively, these data suggest that knockdown of vesicle trafficking gene Gosr2, Arf4, Cog6 and Pacs1 increases mineralized nodule formation in vivo in a P4ha2-dependent manner.

Discussion
By performing RNA-sequencing on three sources of Col2.3GFP + maturing osteoblasts, we identified 593 overlapping genes that are enriched among osteoblasts including genes encoding osteogenic markers (e.g., Ibsp, Bglap), osteogenic transcription factors (e.g., Runx2, Sp7, Dlx5) as well as genes in which mutations have been associated   www.nature.com/scientificreports/with skeletal defects (e.g., Sec24d, Creb3l1).Of note, the top three gene annotation terms were all related to vesicle mediated ER/Golgi transport.We found that transient siRNA knockdown of 15 trafficking genes (Sec24d, Aplp1, Gosr2, Rab2a, Stx5a, Bet1, Preb, Golph3, Golga2, Arf4, Ramp1, Golga4, Cog6, Myo18a and Pacs1) increased mineralized nodule formation and osteoblast marker gene expression.Vesicle trafficking proteins are responsible for cargo selection, transportation, and secretion 2 .Disruption of vesicle trafficking gene expression and function can lead to the unfolded protein response, delayed collagen folding, prolonged collagen transportation, and increased collagen modification.In OI type I less than 50% of collagen is produced in an environment with a normal level of lysyl hydroxylating enzymes which results in an increased enzyme to collagen ratio and subsequent higher levels of hydroxylation and glycosylation as well 14 .Also, proline hydroxylation enhances the stability of the collagen triple helices and lysine modifications increase crosslinking 16 .In OI increased hydroxylation and glycosylation of collagen leads to greater distance between collagen molecules within the fibril.As a result of this steric hinderance more crystals can be deposited within collagen fibrils, leading to hypermineralization 14 .Increased expression of ER stress markers associated with knockdown of Gosr2, Rab2a, Arf4, Cog6, Pacs1, Bet1, Stx5a or Ramp1 suggest that ER homeostasis may have been disrupted.It has been shown that increased matrix deposition during endochondral ossification is associated with activation of endoplasmic reticulum stress sensors, which in turn changed the availability of the metabolic substrates and further increased gene expression levels of collagenmodifying enzymes including collagen prolyl hydroxylases, lysine hydroxylases and lysyl oxidase 15 .Therefore, one possible mechanism for the increased mineralized nodule formation seen with knockdown of trafficking genes Gosr2, Rab2a, Arf4, Cog6, Pacs1, Bet1, Stx5a or Ramp1 might be due to enhanced prolyl 4-hydroxylation caused  Additional collagen-modifying enzymes may also play a role.particular, expression of prolyl 3-hydroxylase genes P3h1 and/or P3h2 was increased by knockdown of Aplp1, Arf4, Myo18a or Pacs1 while expression of the lysine hydroxylase gene Plod was increased by knockdown of Sec24d, Gosr2, Bet1, Arf4 or Pacs1.Of the 15 vesicle trafficking genes we identified that exhibited increased mineralized nodule formation upon transient knockdown, knockdown of Golph3, Golga2 and Golga4 was not associated with increased P4ha, P3h, and Plod expression.Transient knockdown of trafficking genes was also associated with increased expression of osteoblast marker genes, suggesting enhanced osteoblast differentiation.Strikingly, double knockdown P4ha1 or P4ha2 with Gosr2, Rab2a, Stx5a, Bet1, Arf4, Ramp1, Cog6 and Pacs1 did not rescue increased osteoblast marker gene expression, suggesting that regulation of osteoblast differentiation by trafficking genes may be an independent mechanism contributing to mineralized nodule formation.Further investigation and elucidation of signal pathways and mechanisms involved in trafficking gene regulating osteoblast differentiation are important.
The specific function of each trafficking gene in osteoblast differentiation and function remains to be determined.Since siRNA-mediated knockdown is transient, we performed CRISPR/Cas-9-mediated gene editing to permanently knock down Cog6 −/− and Pacs1 −/− in MC3T3-E1 pre-osteoblasts and found that this similarly increases mineralized nodule formation and osteoblast marker gene expression in vitro.
We have further demonstrated that MC3T3 pre-osteoblasts with transient knockdown of Gosr2, Arf4, Cog6 and Pacs1 form more and larger ectopic bone nodules in vivo.However, conditional knockout models will be required to examine the role of trafficking genes in endogenous bone formation, and on bone mass, strength and quality.Cog6 (Component of Oligomeric Golgi Complex 6) belongs to the COG family which is an evolutionally conserved Golgi-associated tethering complex 17 .Cog6 is directly involved in both intra-Golgi and endosome to Golgi retrograde transport 18 .Interestingly, a recent mouse genome-wide association study (GWAS) identified a bone mineral density-associated locus containing Cog6 and showed that Cog6 was highly expressed both in bone and osteoblasts 19 .Pacs1 (Phosphofurin acidic cluster sorting protein 1) is a member of the trans-Golgi network (TGN) membrane protein family and directs the TGN localization of furin 20 .Pacs1 is critical for protein trafficking in vertebrates by binding to diverse cargo and trafficking machinery, including the adaptor protein-1 complex, to ensure the proper cytoplasmic localization of numerous proteins [21][22][23] .A function for Pacs1 in osteoblasts or bone has not been reported.Gosr2 (Golgi SNAP Receptor Complex Member 2) belongs to a group of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are essential for budding, docking and fusion of COPII-coated vesicles during ER-Golgi membrane trafficking 24,25 .Gosr2 gene was identified as a causative gene for progressive myoclonus epilepsy with distinct clinical features include scoliosis 26 .It has also been shown that Gosr2 forms complex with Stx5 and Bet1 and the complex are responsible for fusion of endoplasmic reticulum derived vesicles with the ER-Golgi intermediate compartment and the cis-Golgi 24,25,27 .Similar to knockdown of Gosr2, we also found increased osteogenic differentiation and mineralized nodule formation with knockdown of Stx5, Bet1 and Rab2a in MC3T3-E1 pre-osteoblasts.Both Bet1 and Rab2a are members of the GTPase family and are involved in post-endocytic trafficking of membrane-bound MT1-MMP (MMP14, membrane type 1-matrix metalloproteinase), an essential metalloprotease for matrix remodeling, invasion, pericellular collagenolysis and skeletal/extraskeletal connective tissue modeling [28][29][30] .Arf4 (ADP Ribosylation Factor 4) is another vesicle trafficking protein belonging to GTPase family and functions an ADP-ribosyltransferase 31 .ARF4 regulates ciliary protein trafficking, dysfunction of which is a known cause of human genetic diseases and syndromic disorders known as ciliopathies [31][32][33] .Both Rab2a and Arf4 are highly expressed in osteoblasts and bone (data from BioGPS, http:// biogps.org/) 34 .
In summary, we identified a set of vesicle trafficking genes that are highly expressed in Col2.3GFP + osteoblast lineage cells and play a role in osteoblast differentiation and mineralized nodule formation.Osteoblasts synthesize, transport, and secrete large amounts of bone matrix components including collagen, which is tightly regulated.Any dysregulation would cause inadequate or excessive mineralization of bones.Elucidation of the specific function of vesicle traffic genes involving in osteoblasts function raises the possibility of developing novel therapeutics for bone diseases designed to target this process.

Tissue culture and osteogenic differentiation
Generation, maintenance and differentiation of mouse Col2.3GFPESCs were performed as previously described 9 .Bone chips were harvested from Col2.3GFP transgenic mice and subjected to osteogenic differentiation as previously described 35 .

RNA interference
For transient knockdown target gene, siGENOME SMARTpool siRNA for specific target genes and non-Targeting siRNA for siControl were synthesized and purchased from Dharmacon, Inc. (Lafayette, CO).Cells were plated at a concentration of 1 X 10 4 cells/well in 24-well plates and transfected with the siRNA using Lipofectamine RNAiMax transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions on the following day after plating.2.5 µl of 10 µM siRNA duplexes were mixed with 47.5 µl Opti-MEM (Thermo Fisher Scientific) and combined with 1 µl/well of transfection reagent and 49 µl Opti-MEM to a total volume of 100 µl and incubated for 20 min.After incubation, 100 µl siRNA-lipid complex were transfected to the adherent cells.Osteogenic differentiation was initiated with osteogenic medium on the second day after transfection.Transfected cells were analyzed at different time points.

Von Kossa, Alizarin Red S and alkaline phosphatase staining
For Von Kossa staining, cells were rinsed twice with PBS and fixed in 10% formaldehyde for 10 min, then washed with double distilled water (DDW).Cells were incubated in 5% silver nitrate solution under UV light for 30 min, washed with DDW, rinsed with 5% sodium thiosulfate for 5 min to remove unreacted silver, washed again with DDW and stored in PBS.For Alizarin Red S staining cells were fixed and washed, stained with 2% Alizarin Red S solution for 20 min, then washed with DDW to remove unincorporated excess dye.For quantification of Alizarin Red S staining, following the wash in DDW cells were incubated in 10% Acetic acid with shaking for 30 min at room temperature.Cells were collected by cell scraper and transferred to microcentrifuge tubes, heated for 10 min at 85 °C, and then put on ice for 5 min.After centrifugation at 20,000 g for 15 min the supernatant was transferred to a new microcentrifuge tube.Upon addition of 10% Ammonium hydroxide, 50 µl aliquot was transferred to a microplate reader well and absorbance was measured at 405 nm.Alkaline phosphatase staining was performed by using Stemgent ® Alkaline Phosphatase Staining Kit II (Stemgent, San Diego, CA) according to the manufacturer's instructions.

Quantitative reverse transcription PCR (qPCR) and RNA-sequencing
RNA was extracted by using PureLink ® RNA Mini Kit (Invitrogen, Carlsbad, CA).RNA concentration and purity were measured by NanoDrop (Thermo Scientific, Wilmington, DE) and then used for reverse transcription using iScript Kit (Bio-Rad, Hercules, CA).Quantitative RT-PCR (qPCR) was performed following standard methods.Primer sequences are provided in Supplementary Table.mRNA levels were normalized to of the levels of β-Actin.
Relative mRNA levels of trafficking gene knockdown cells were then normalized to siControl.PCR was performed in triplicate for each sample, and 3 independent experiments were carried out 11 .
For RNA-sequencing (RNA-seq), the integrity of extracted RNA was assayed by on-chip electrophoresis (Agilent Bioanalyzer) and only samples with a high RNA integrity (RIN) value were used for RNA-seq 11 .Poly-A mRNA was purified using Dynabeads ® mRNA Purification Kit (Invitrogen) according to the manufacturer's instructions.RNA sequencing libraries were prepared using the NEBNext ® Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions.Library quality was verified using the Agilent High Sensitivity DNA Kit on Agilent's 2100 Bioanalyzer.For each library, an average of 420 bp fragments were sequenced using paired end reads (2 × 100 bp) on the Illumina HiSeq 2500 platform (Stanford Personalized Medicine Sequencing Core), with an average of 30 million reads per sample.Paired end sequencing reads (100 bp) were generated and aligned to mouse reference sequence NCBI Build 37/mm9 with the STAR (v2.4.2a) algorithm 36 .Normalization of RefSeq annotated genes, expression level and differential expression analysis were performed using the Bioconductor package DESeq2 in R (Version 3.2.2) 37 .Genes with False Discovery Rate (FDR) < 0.05 were defined as differentially expressed genes.Gene ontology analysis was performed using DAVID (david.abcc.ncifcrf.gov)and The Gene Ontology (GO) Consortium (http:// geneo ntolo gy.org/).For Gene Set Enrichment Analysis (GSEA, Broad Institute), gene set associated with osteogenesis were obtained from www. qiagen.com (Qiagen, Germany) 38 .All transcriptome raw data are publicly available in GEO (accession number GSE223192, Genome wide gene expression profiling of Col2.3GFP + enriched osteoblasts).

Subcutaneous implantation
Seven-week-old male CD-1 Nude Mice Crl:CD1-Foxn1nu purchased from Charles River (Wilmington, MA, USA) were housed in Innovive recyclable individually ventilated cages in a designated pathogen-free area facility and fed irradiated mouse chow and autoclaved water.The Veterinary Service Center (VSC) at Stanford University provides laboratory animal care and is administered by the Department of Comparative Medicine.The laboratory animal care program at Stanford University is fully accredited by the Association for Accreditation and Assessment of Laboratory Animal Care (AAALAC).All animal surgeries were approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC).All methods were performed in accordance with

Figure 1 .Figure 2 .
Figure 1.Genes involved in vesicle trafficking are enriched in transcriptomes of mature osteoblasts.(A) Gene set enrichment analysis (GSEA) shows significantly upregulated genes in Col2.3GFP + population are enriched in osteogenesis gene set.(B) 593 overlapping genes that highly enriched in Col2.3GFP + osteoblasts from all three resources are identified.(C) Gene Ontology Analysis shows that the top 3 annotation terms are all associated with ER and Golgi vesicle transport.

Figure 7 .
Figure 7. Vesicle trafficking gene depletion increases mineralized nodule formation in vivo.MC3T3-E1 cells transfected with Control siRNA, trafficking gene siRNA with/without P4ha2 siRNA and Matrigel mixtures were subcutaneously injected into immunodeficient mice.Eight weeks later, mice were euthanized and dissected.(A) Representative micro-CT X-ray images of explanted nodules show significant mineralized structure in each cell group of implants.(B) Incidences of mineralized nodule formation in each group.*P < 0.05; Z proportion score.(C) Quantification of mineralized nodule volume and micro-CT total intensity in each group.All data represent with interleaved scatter plots with mean ± SD (multiple t test; *P < 0.05; **P < 0.01; # P < 0.001).(D) Serial cryosections were made in each explanted cell nodules.Mineralized structures were visualized using Alizarin red S and Von Kossa staining.Newly formed mineralized structures were visualized by Aniline Blue staining.