PTH Regulates β2‐Adrenergic Receptor Expression in Osteoblast‐Like MC3T3‐E1 Cells

Moriya, Shuichi; Hayata, Tadayoshi; Notomi, Takuya; Aryal, Smriti A C; Nakamaoto, Testuya; Izu, Yayoi; Kawasaki, Masanori; Yamada, Takayuki; Shirakawa, Jumpei; Ezura, Yoichi; Noda, Masaki

doi:10.1002/jcb.24953

Cited by 16 publications

(14 citation statements)

References 33 publications

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“…2 . Studies using MC3T3-E1 cells to examine the role of signaling through the parathyroid hormone receptor (PTH1R) in bone metabolism have been particularly confounding 4–7 . It is possible that this incongruity may be due to heterogeneity of the parent cell line and subsequent phenotypic divergence of its derivative subclones.…”

Section: Introductionmentioning

confidence: 99%

Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology

Hwang

Horton

2019

Sci Rep

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The spontaneously immortalized murine calvarial cell line MC3T3-E1 and its derivative subclones are widely used models of osteoblast biology. Many investigators have reported conflicting data under seemingly similar experimental conditions, though the specific subclone studied is often not specified. The purpose of this study was to directly compare the commercially available MC3T3-E1 subclones 4, 14, and 24 in terms of responsiveness to osteogenic induction media and/or stimulation with rhPTH[1–34]. We assayed osteogenic gene expression, capacity to deposit and mineralize a collagenous matrix, and the expression and signaling function of PTH1R. Our data demonstrate that each subclone bears little functional resemblance to the others, or to primary calvarial osteoblasts. Specifically, whereas subclone 4 is responsive to PTH stimulation and capable of matrix mineralization, subclones 14 and 24 do not faithfully replicate these key aspects of osteoblast biology. Furthermore, little overlap was observed between the gene expression profile of subclone 4 and primary calvarial osteoblasts. Our experience working with these cell lines demonstrates that the MC3T3-E1 derived cell lines are imperfect models of osteoblast biology, and reinforce the importance of clearly articulating selection and reporting of research materials.

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Section: Introductionmentioning

confidence: 99%

Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology

Hwang

Horton

2019

Sci Rep

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“…Although a number of efficient therapeutic agents are available now [3], parathyroid hormone (PTH1-84) or the synthetic peptide PTH1-34 (teriparatide) recapitulating the effects of PTH is the only agent that induces anabolic action of bone formation [4,5]. Molecular mechanisms of the anabolic action of PTH have been investigated from various aspects [6][7][8]. For example, PTH1-34 suppresses sclerostin gene (Sost) expression in osteocytes, and thus increases osteoblastic bone formation through activation of Wnt-Lrp5/6 to b-Catenin signaling pathway [9,10].…”

Section: Introductionmentioning

confidence: 99%

PTH-Induced Osteoblast Proliferation Requires Upregulation of the Ubiquitin-Specific Peptidase 2 (Usp2) Expression

et al. 2015

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Osteoporosis is a common disease that increases individual's fragility fracture risk. PTH is the only therapeutic agent for severe osteoporosis that requires anabolic action of bone formation. Although a part of the PTH actions is explained by increased proliferation of osteoblastic precursor cells, the mechanisms involved in the proliferation of osteoblastic cells by PTH have not been clarified yet. Therefore, in this study, we investigated the effects of PTH on gene expression in the cultured osteoblastic MC3T3-E1 cells, and found that the ubiquitin-specific peptidase 2 (Usp2) may be one of the direct target genes of PTHR signaling. Usp2 is a deubiquitination enzyme targeting various factors including CyclinD1 in cancer cells and PTH receptor 1 in osteoblasts. We confirmed that consistent induction of Usp2 expression peaked at 1 h by PTH1-34 (teriparatide) in MC3T3-E1 cells and primary calvarial osteoblasts. Among the three known splicing variants of the Usp2, we found the isoforms 1 and 2 are predominantly expressed in osteoblasts. Live-imaging analysis of the Fucci-transgenic mouse-derived primary osteoblasts indeed demonstrated that Usp2 is required for the PTH1-34-induced osteoblast proliferation. Western blotting analysis of the CyclinD1 indicated that Usp2 knock-down influences the paradoxical changes of CyclinD1 protein levels in this condition. Our data indicate that Usp2 is required for the PTH1-34-induced proliferation of osteoblasts.

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“…In light of the observations that VPS35 regulates PTH1R's trafficking and signaling in HEK293 cells ( Feinstein et al, 2011 ), and both VPS35 and PTH1R are highly expressed in OBs ( Simmonds and Kovacs, 2010 , Vilardaga et al, 2012 , Xia et al, 2013b ), we speculate a crucial role for VPS35 to play in regulating PTH1R's trafficking, signaling, and function in OB-lineage cells. To test this speculation, we first examined exogenous PTH1R's trafficking in control and Vps35-deficient MC3T3 cells, an osteoblastic cell line that responses to PTH (1–34) stimulation ( Issack et al, 2007 , Moriya et al, 2015 , Schiller et al, 1999 ). A plasmid encoding PTH1R-mCherry fusion protein was generated, which is a similar fusion protein as PTH1R-EGFP that is often used in the literature to image PTH1R's distribution ( Feinstein et al, 2011 ) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Retromer in Osteoblasts Interacts With Protein Phosphatase 1 Regulator Subunit 14C, Terminates Parathyroid Hormone's Signaling, and Promotes Its Catabolic Response

et al. 2016

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Parathyroid hormone (PTH) plays critical, but distinct, roles in bone remodeling, including bone formation (anabolic response) and resorption (catabolic response). Although its signaling and function have been extensively investigated, it just began to be understood how distinct functions are induced by PTH activating a common receptor, the PTH type 1 receptor (PTH1R), and how PTH1R signaling is terminated. Here, we provide evidence for vacuolar protein sorting 35 (VPS35), a major component of retromer, in regulating PTH1R trafficking, turning off PTH signaling, and promoting its catabolic function. VPS35 is expressed in osteoblast (OB)-lineage cells. VPS35-deficiency in OBs impaired PTH(1–34)-promoted PTH1R translocation to the trans-Golgi network, enhanced PTH(1–34)-driven signaling, and reduced PTH(1–34)'s catabolic response in culture and in mice. Further mechanical studies revealed that VPS35 interacts with not only PTH1R, but also protein phosphatase 1 regulatory subunit 14C (PPP1R14C), an inhibitory subunit of PP1 phosphatase. PPP1R14C also interacts with PTH1R, which is necessary for the increased endosomal PTH1R signaling and decreased PTH(1–34)'s catabolic response in VPS35-deficient OB-lineage cells. Taken together, these results suggest that VPS35 deregulates PTH1R-signaling likely by its interaction with PTH1R and PPP1R14C. This event is critical for the control of PTH(1–34)-signaling dynamics, which may underlie PTH-induced catabolic response and adequate bone remodeling.

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PTH Regulates β2‐Adrenergic Receptor Expression in Osteoblast‐Like MC3T3‐E1 Cells

Cited by 16 publications

References 33 publications

Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology

Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology

PTH-Induced Osteoblast Proliferation Requires Upregulation of the Ubiquitin-Specific Peptidase 2 (Usp2) Expression

Retromer in Osteoblasts Interacts With Protein Phosphatase 1 Regulator Subunit 14C, Terminates Parathyroid Hormone's Signaling, and Promotes Its Catabolic Response

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