Silencing Itch in human peripheral blood monocytes promotes their differentiation into osteoclasts

Oliver J. Read; David J. Harrison

doi:10.1007/s11033-022-07726-1

Silencing Itch in human peripheral blood monocytes promotes their differentiation into osteoclasts

Oliver J. Read^*, David J. Harrison

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

ntroduction

Two clinical case reports of humans with mutations in Itch reported distinct morphological defects such as stunted growth, macrocephaly, and dysmorphic features indicating a role for Itch in bone remodelling. Studies in mice have found that the encoded E3 ubiquitin ligase acts as a negative regulator of osteoclastogenesis, however no studies have investigated whether this is translatable to a human model.

Experimental procedures

Human peripheral blood monocytes were separated from whole blood and grown in M-CSF containing media. Media was later supplemented with RANKL to promote osteoclast differentiation. Transient siRNA-mediated Itch knockdown (si-Itch) in monocytes was verified by qPCR and western blot to confirm reduction in both Itch mRNA and protein respectively. Monocytes were aliquoted onto 96-well plates where confluence and osteoclast formation were analysed using automated cytometry analysis before and after staining for tartrate resistant acid phosphatase activity (TRAP). Cells were also stained with Hoechst33342 to look for multinucleate cells.

Results

Cells treated with si-Itch showed an 80% knockdown in Itch mRNA and > 75% reduction in protein. Following the 7-day differentiation period, si-Itch caused a 47% increase in multinucleate cells and a 17% increase in numbers of large cellular bodies and, indicating an overall increase in mature osteoclast formation.

Conclusions

Our preliminary data shows silencing Itch expression increases the potential of primary human monocytes to differentiate into osteoclast-like cells in vitro.

Original language	English
Journal	Molecular Biology Reports
Volume	First Online
Early online date	6 Jul 2022
DOIs	https://doi.org/10.1007/s11033-022-07726-1
Publication status	E-pub ahead of print - 6 Jul 2022

Keywords

Itch
Monocyte
Osteoclast
E3 ubiquitin ligase
Knockdown

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Cite this

@article{27b0ce5107a24c748820598ec263d79c,

title = "Silencing Itch in human peripheral blood monocytes promotes their differentiation into osteoclasts",

abstract = "ntroductionTwo clinical case reports of humans with mutations in Itch reported distinct morphological defects such as stunted growth, macrocephaly, and dysmorphic features indicating a role for Itch in bone remodelling. Studies in mice have found that the encoded E3 ubiquitin ligase acts as a negative regulator of osteoclastogenesis, however no studies have investigated whether this is translatable to a human model.Experimental proceduresHuman peripheral blood monocytes were separated from whole blood and grown in M-CSF containing media. Media was later supplemented with RANKL to promote osteoclast differentiation. Transient siRNA-mediated Itch knockdown (si-Itch) in monocytes was verified by qPCR and western blot to confirm reduction in both Itch mRNA and protein respectively. Monocytes were aliquoted onto 96-well plates where confluence and osteoclast formation were analysed using automated cytometry analysis before and after staining for tartrate resistant acid phosphatase activity (TRAP). Cells were also stained with Hoechst33342 to look for multinucleate cells.ResultsCells treated with si-Itch showed an 80% knockdown in Itch mRNA and > 75% reduction in protein. Following the 7-day differentiation period, si-Itch caused a 47% increase in multinucleate cells and a 17% increase in numbers of large cellular bodies and, indicating an overall increase in mature osteoclast formation.ConclusionsOur preliminary data shows silencing Itch expression increases the potential of primary human monocytes to differentiate into osteoclast-like cells in vitro.",

keywords = "Itch, Monocyte, Osteoclast, E3 ubiquitin ligase, Knockdown",

author = "Read, {Oliver J.} and Harrison, {David J.}",

note = "Funding: This work for funded by the School of Medicine, University of St Andrews.",

year = "2022",

month = jul,

day = "6",

doi = "10.1007/s11033-022-07726-1",

language = "English",

volume = "First Online",

journal = "Molecular Biology Reports",

issn = "1573-4978",

publisher = "Springer",

}

TY - JOUR

T1 - Silencing Itch in human peripheral blood monocytes promotes their differentiation into osteoclasts

AU - Read, Oliver J.

AU - Harrison, David J.

N1 - Funding: This work for funded by the School of Medicine, University of St Andrews.

PY - 2022/7/6

Y1 - 2022/7/6

N2 - ntroductionTwo clinical case reports of humans with mutations in Itch reported distinct morphological defects such as stunted growth, macrocephaly, and dysmorphic features indicating a role for Itch in bone remodelling. Studies in mice have found that the encoded E3 ubiquitin ligase acts as a negative regulator of osteoclastogenesis, however no studies have investigated whether this is translatable to a human model.Experimental proceduresHuman peripheral blood monocytes were separated from whole blood and grown in M-CSF containing media. Media was later supplemented with RANKL to promote osteoclast differentiation. Transient siRNA-mediated Itch knockdown (si-Itch) in monocytes was verified by qPCR and western blot to confirm reduction in both Itch mRNA and protein respectively. Monocytes were aliquoted onto 96-well plates where confluence and osteoclast formation were analysed using automated cytometry analysis before and after staining for tartrate resistant acid phosphatase activity (TRAP). Cells were also stained with Hoechst33342 to look for multinucleate cells.ResultsCells treated with si-Itch showed an 80% knockdown in Itch mRNA and > 75% reduction in protein. Following the 7-day differentiation period, si-Itch caused a 47% increase in multinucleate cells and a 17% increase in numbers of large cellular bodies and, indicating an overall increase in mature osteoclast formation.ConclusionsOur preliminary data shows silencing Itch expression increases the potential of primary human monocytes to differentiate into osteoclast-like cells in vitro.

AB - ntroductionTwo clinical case reports of humans with mutations in Itch reported distinct morphological defects such as stunted growth, macrocephaly, and dysmorphic features indicating a role for Itch in bone remodelling. Studies in mice have found that the encoded E3 ubiquitin ligase acts as a negative regulator of osteoclastogenesis, however no studies have investigated whether this is translatable to a human model.Experimental proceduresHuman peripheral blood monocytes were separated from whole blood and grown in M-CSF containing media. Media was later supplemented with RANKL to promote osteoclast differentiation. Transient siRNA-mediated Itch knockdown (si-Itch) in monocytes was verified by qPCR and western blot to confirm reduction in both Itch mRNA and protein respectively. Monocytes were aliquoted onto 96-well plates where confluence and osteoclast formation were analysed using automated cytometry analysis before and after staining for tartrate resistant acid phosphatase activity (TRAP). Cells were also stained with Hoechst33342 to look for multinucleate cells.ResultsCells treated with si-Itch showed an 80% knockdown in Itch mRNA and > 75% reduction in protein. Following the 7-day differentiation period, si-Itch caused a 47% increase in multinucleate cells and a 17% increase in numbers of large cellular bodies and, indicating an overall increase in mature osteoclast formation.ConclusionsOur preliminary data shows silencing Itch expression increases the potential of primary human monocytes to differentiate into osteoclast-like cells in vitro.

KW - Itch

KW - Monocyte

KW - Osteoclast

KW - E3 ubiquitin ligase

KW - Knockdown

U2 - 10.1007/s11033-022-07726-1

DO - 10.1007/s11033-022-07726-1

M3 - Article

SN - 1573-4978

VL - First Online

JO - Molecular Biology Reports

JF - Molecular Biology Reports

ER -

Silencing Itch in human peripheral blood monocytes promotes their differentiation into osteoclasts

Abstract

Keywords

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