Rab GTPase Mediated Procollagen Trafficking in Ascorbic Acid Stimulated Osteoblasts

Nabavi, Noushin; Pustylnik, Sofia; Harrison, Rene E.

doi:10.1371/journal.pone.0046265

Cited by 18 publications

(20 citation statements)

References 73 publications

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“…We compared our list of differentially expressed genes with a previously published expression profile from MC3T3-E1 calvarial pre-osteoblasts 5 days following induction of osteogenic differentiation (GSE37676)[37]. Using an FDR of 5%, the two datasets shared 442 differentially expressed genes (Figure 5A).…”

Section: Resultsmentioning

confidence: 99%

IL15RA is required for osteoblast function and bone mineralization

Loro

Ramaswamy

Chandra

et al. 2017

Bone

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Interleukin-15 receptor alpha (IL15RA) is an important component of interleukin-15 (IL15) pro-inflammatory signaling. In addition, IL15 and IL15RA are present in the circulation and are detected in a variety of tissues where they influence physiological functions such as muscle contractility and overall metabolism. In the skeletal system, IL15RA was previously shown to be important for osteoclastogenesis. Little is known, however, about its role in osteoblast function and bone mineralization. In this study, we evaluated bone structural and mechanical properties of an Il15ra whole-body knockout mouse (Il15ra−/−) and used in vitro and bioinformatic analyses to understand the role IL15/IL15RA signaling on osteoblast function. We show that lack of IL15RA decreased bone mineralization in vivo and in isolated primary osteogenic cultures, suggesting a cell-autonomous effect. Il15ra−/− osteogenic cultures also had reduced Rankl/Opg mRNA ratio, indicating defective osteoblast/osteoclast coupling. We analyzed the transcriptome of primary pre-osteoblasts from normal and Il15ra−/− mice and identified 1150 genes that were differentially expressed at a FDR of 5%. Of these, 844 transcripts were upregulated and 306 were downregulated in Il15ra−/− cells. The largest functional clusters, highlighted using DAVID analysis, were related to metabolism, immune response, bone mineralization and morphogenesis. The transcriptome analysis was validated by qPCR of some of the most significant hits. Using bioinformatic approaches, we identified candidate genes, including Cd200 and Enpp1, that could contribute to the reduced mineralization. Silencing Il15ra using shRNA in the calvarial osteoblast MC3T3-E1 cell line decreased ENPP1 activity. Taken together, these data support that IL15RA plays a cell-autonomous role in osteoblast function and bone mineralization.

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Section: Resultsmentioning

confidence: 99%

IL15RA is required for osteoblast function and bone mineralization

Loro

Ramaswamy

Chandra

et al. 2017

Bone

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“…recently performed a microarray analysis to identify the up-regulated Rab GTPases that are important for procollagen trafficking during osteoblast differentiation (40). Our microarray analysis revealed an expected up-regulation of the components of the Wnt signaling pathway (Fig.…”

Section: Eb1 Is Up-regulated In Aa-stimulated Osteoblasts-wementioning

confidence: 87%

“…40. MC3T3-E1 osteoblasts were grown in 6-well plates in triplicates and differentiated with AA (50 g/ml) for 5 days.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA from control and AA-treated osteoblasts was purified from cultured cells using the RNeasy minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Samples were hybridized to MOE430.20 GeneChips (Affymetrix, Santa Clara, CA) at the Center for Applied Genomics at SickKids (Toronto, Canada) and analyzed as described previously (40). -Fold changes in gene expression levels were analyzed using Student's unpaired t tests.…”

Section: Methodsmentioning

confidence: 99%

“…A critical p value of 0.01 was considered as the criterion to select a significant -fold change in gene expression. Of the total 45,103 genes identified by (40), EB1 and components of Wnt signaling were selected for further investigation in the present study.…”

Section: Methodsmentioning

confidence: 99%

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EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation

Pustylnik

Fiorino

Nabavi

et al. 2013

Journal of Biological Chemistry

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Background: EB1 is a microtubule (MT) plus-end-binding protein known to influence MT stability. Results: EB1 is up-regulated in osteoblasts and is required for bone cell differentiation. Conclusion: EB1 affects ␤-catenin stability and cooperates at cell-cell adhesion sites to influence gene expression. Significance: Learning how peripherally targeted proteins interact at cell-cell contact sites is crucial for understanding developmental processes.

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Traf2 and NCK Interacting Kinase Is a Critical Regulator of Procollagen I Trafficking and Hepatic Fibrogenesis in Mice

et al. 2021

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Hepatic fibrosis is driven by deposition of matrix proteins following liver injury. Hepatic stellate cells (HSCs) drive fibrogenesis, producing matrix proteins, including procollagen I, which matures into collagen I following secretion. Disrupting intracellular procollagen processing and trafficking causes endoplasmic reticulum stress and stress-induced HSC apoptosis and thus is an attractive antifibrotic strategy. We designed an immunofluorescence-based small interfering RNA (siRNA) screen to identify procollagen I trafficking regulators, hypothesizing that these proteins could serve as antifibrotic targets. A targeted siRNA screen was performed using immunofluorescence to detect changes in intracellular procollagen I. Tumor necrosis factor receptor associated factor 2 and noncatalytic region of tyrosine kinase-interacting kinase (TNIK) was identified and interrogated in vitro and in vivo using the TNIK kinase inhibitor NCB-0846 or RNA interference-mediated knockdown. Our siRNA screen identified nine genes whose knockdown promoted procollagen I retention, including the serine/threonine kinase TNIK. Genetic deletion or pharmacologic inhibition of TNIK through the small molecule inhibitor NCB-0846 disrupted procollagen I trafficking and secretion without impacting procollagen I expression. To investigate the role of TNIK in liver fibrogenesis, we analyzed human and murine livers, finding elevated TNIK expression in human cirrhotic livers and increased TNIK expression and kinase activity in both fibrotic mouse livers and activated primary human HSCs. Finally, we tested whether inhibition of TNIK kinase activity could limit fibrogenesis in vivo. Mice receiving NCB-0846 displayed reduced CCl 4 -induced fibrogenesis compared to CCl 4 alone, although α-smooth muscle actin levels were unaltered. Conclusions: Our siRNA screen effectively identified TNIK as a key kinase involved in procollagen I trafficking in vitro and hepatic fibrogenesis in vivo. (Hepatology Communications 2022;6:593-609).

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Rab GTPase Mediated Procollagen Trafficking in Ascorbic Acid Stimulated Osteoblasts

Cited by 18 publications

References 73 publications

IL15RA is required for osteoblast function and bone mineralization

IL15RA is required for osteoblast function and bone mineralization

EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation

Traf2 and NCK Interacting Kinase Is a Critical Regulator of Procollagen I Trafficking and Hepatic Fibrogenesis in Mice

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