Name: stimulation of notch signaling in mouse osteoclast precursors
Uploaded: 2017-02-28T15:00:00
Description: Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

This work was supported by the University of Pennsylvania Center for Musculoskeletal Disorders (5 P30 AR050950-09), a grant from the AO Foundation (S-16-12A), the Philadelphia VA Medical Center Translational Musculoskeletal Research Center, and an intramural orthopaedic surgery departmental research development fund. JWA is supported by the University of Pennsylvania Postdoctoral Opportunities in Research and Teaching (PENN-PORT) fellowship funded by the National Institute of General Medical Sciences Institutional Research and Career Development Award (IRACDA; 5 K12 GM081259-08).

Toda a investigação que envolva animais vertebrados foi realizada de acordo com protocolos aprovados pela Universidade da Pensilvânia Animal Care Institucional e Comitê de Uso (IACUC). 1. Meios de Cultura Preparação <ol><li> Prepare α-MEM por dissolução médio (MEM) em pó essencial mínimo e 1,9 g de bicarbonato de sódio em 900 ml de H 2 O e completar com 2 mM de L-glutamina, 100 unidades / ml de penicilina, 100 ug / ml de estreptomicina, e 100 mL de calor de soro bov...

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D., Shimada, I. S., Sherwin, C., Spees, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1-STAT3-ETBR+signaling+axis+controls+reactive+astrocyte+proliferation+after+brain+injury.">Notch1-STAT3-ETBR signaling axis controls reactive astrocyte proliferation after brain injury.</a> Proc Natl Acad Sci U S A. 112 (28), 8726-8731 (2015).</li><li>Kostianovsky, A. M., Maier, L. M., Baecher-Allan, C., Anderson, A. C., Anderson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+gene+related+to+anergy+in+lymphocytes+is+associated+with+Notch-mediated+human+T+cell+suppression.">Up-regulation of gene related to anergy in lymphocytes is associated with Notch-mediated human T cell suppression.</a> J Immunol. 178 (10), 6158-6163 (2007).</li><li>Teitelbaum, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclasts:+what+do+they+do+and+how+do+they+do+it?.">Osteoclasts: what do they do and how do they do it?.</a> Am J Pathol. 170 (2), 427-435 (2007).</li><li>Ma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+transcription+factor+RBP-J+results+in+osteopenia+attributable+to+attenuated+osteoclast+differentiation.">Disruption of the transcription factor RBP-J results in osteopenia attributable to attenuated osteoclast differentiation.</a> Mol Biol Rep. 40 (3), 2097-2105 (2013).</li><li>Bai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOTCH1+regulates+osteoclastogenesis+directly+in+osteoclast+precursors+and+indirectly+via+osteoblast+lineage+cells.">NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells.</a> J Biol Chem. 283 (10), 6509-6518 (2008).</li><li>Yamada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+osteoclast+development+by+Notch+signaling+directed+to+osteoclast+precursors+and+through+stromal+cells.">Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells.</a> Blood. 101 (6), 2227-2234 (2003).</li><li>Sethi, N., Dai, X., Winter, C. G., Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+JAGGED1+promotes+osteolytic+bone+metastasis+of+breast+cancer+by+engaging+notch+signaling+in+bone+cells.">Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.</a> Cancer Cell. 19 (2), 192-205 (2011).</li><li>Sekine, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+osteoclastogenesis+by+Notch2/Delta-like+1+and+Notch1/Jagged1+axes.">Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes.</a> Arthritis Res Ther. 14 (2), R45 (2012).</li><li>Ashley, J. W., Ahn, J., Hankenson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signaling+Promotes+Osteoclast+Maturation+and+Resorptive+Activity.">Notch Signaling Promotes Osteoclast Maturation and Resorptive Activity.</a> J Cell Biochem. 116 (11), 2598-2609 (2015).</li><li>Fukushima, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+of+Notch2+and+NF-kappaB+accelerates+RANKL-induced+osteoclastogenesis.">The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis.</a> Mol Cell Biol. 28 (20), 6402-6412 (2008).</li><li>Canalis, E., Schilling, L., Yee, S. P., Lee, S. 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As etapas críticas no âmbito do protocolo Cultura e na diferenciação in vitro de precursores de osteoclastos fornece uma plataforma útil para a investigação dos mecanismos moleculares de osteoclastogénese e identificação de alvos terapêuticos para regeneração óssea e preservação da massa óssea. Quando a cultura de precursores de osteoclastos de rato, o elemento mais crítico é a manutenção dos precursores em um estado ingênuo. Como as células de macrófag...

A via de sinalização Notch de mamífero é homóloga da mesma via em Drosophila melanogaster e consiste de quatro receptores transmembrana entalhe (Notch1-4) e cinco ligandos ligados à membrana do entalhado (JAG1 &amp; JAG2) e semelhante a delta (DLL1, 3, &amp; 4) famílias 1. Sinalização Notch é iniciado quando o receptor Notch em uma célula receptora é obrigado pelo ligando em uma célula de transmissão 2. Durante este trans-activação, o ligando ligado à membrana Notch produz uma força de estiramento sobre o receptor de Notch ligada a membrana 3, 4. A força de estiramento do ligando de ligação induz alterações conformacionais no receptor de Notch que facilitam a clivagem extracelular do receptor por TNFalfa enzima de conversão (TACE), seguido por um evento de clivagem intracelular mediada por um complexo de gama-secretase (γ-secretase) contendo presenilina. γ-secretase libera o int Notchdomínio racellular (NICD), que transloca para o núcleo, onde forma um complexo de activação da transcrição com CBF-1-Su (H) -Lag-1 (CSL), idealizador-like (MAML), e fatores específicos do tipo de célula para conduzir a expressão de alvejar genes 5. Os elementos mecânicos de resultado de activação de sinalização Notch na necessidade de métodos exclusivos da activação da via de Notch in vitro. Notch ligandos solúveis podem ligar-se a receptores de Notch, mas não conseguem produzir forças de estiramento necessárias para a libertação de NICD enquanto ao mesmo tempo inibir competitivamente a ligação de ligandos de entalhe associada a células. Assim, a adição de ligantes Notch solúvel para o meio de cultura pode atenuar Notch normal de sinalização 6, 7. Felizmente, os ligandos de Notch pode induzir a libertação de NICD se eles são fixados a um substrato apropriadamente rígida 5, 8, 9,10. Sementeira em células de cultura de substratos revestidos com ligando ou a aplicação de contas revestidas com ligando às células pode tanto activar a sinalização de Notch, e a escolha entre elas depende primeiramente do tempo desejado de estimulação de Notch. Para a activação de sinalização de Notch imediato e temporário, tal como seria desejado durante o ponto médio de um ensaio funcional ou diferenciação, ligando de Notch pode ser ligado a esferas de agarose, aplicados a células de cultura, e lavou-se a qualquer momento. Para mais sinalização Notch sustentada a partir do início de um período de cultura, placas de cultura de tecidos podem ser revestidos com ligando antes da sementeira celular. Para os efeitos do presente protocolo, os métodos são realizados utilizando precursores de osteoclastos de rato, mas os métodos e variações sobre os métodos aqui descritos são aplicáveis a uma ampla variedade de tipos de células 6, 11, 12, 13, </sup&gt; 14. Os osteoclastos são células terminalmente diferenciadas que linage hematopoiéticos são responsáveis pela reabsorção de tecido ósseo, e eles estão implicados em vários distúrbios de perda de osso 15. Assim, no estudo in vitro da diferenciação de osteoclastos a partir dos seus precursores de monócitos / macrófagos de linhagem e os mecanismos moleculares que controlam a sua função é essencial para uma melhor compreensão dos osteoclastos e desenvolvimento de novas terapias de regeneração de osso. Enquanto ele é agora geralmente aceite que a sinalização Notch desempenha um papel positivo na diferenciação e função dos osteoclastos, as variações tanto da estimulação sinalização Notch e osteoclastos cultura precursor e diferenciação conduziu a resultados contraditórios inicialmente 16, 17, 18, 19. Um exame mais detalhado das diferenças de métodos e utilização de genéticamodelos têm muito esclarecido o papel da sinalização Notch na osteoclastogênese, mas a aplicação de métodos de estimulação Notch e cultura padronizados poderia evitar tais controvérsias em estudos futuros de sinalização Notch em outros tipos de células 20, 21, 22, 23. Existem vários métodos para a cultura de precursores e de diferenciação de osteoclastos de rato, e, como com os métodos diferentes para estimular a sinalização de Notch, o melhor método irá depender da questão experimental. Aqui, será apresentado o nosso método preferido de cultura frações aderentes e não aderentes de células de medula liberadas a partir de ossos longos de rato. Este método tem a vantagem de exigir essencialmente nenhum equipamento especializado e a produção de células que são aplicáveis ​​a uma variedade de métodos de diferenciação.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Recombinant mouse M-CSF</td>
			<td>Biolegend</td>
			<td>576402</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Recombinant mouse RANKL</td>
			<td>Shenandoah Biotechnology</td>
			<td>200-04</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Recombinant human Jagged1-Fc</td>
			<td>R&#38;D Systems</td>
			<td>1277-JG-050</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Protein G agarose beads</td>
			<td>InvivoGen</td>
			<td>gel-agg-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG Fc</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-001-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium powder</td>
			<td>Sigma-Aldrich</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase cell dissociation reagent</td>
			<td>ThermoFisher</td>
			<td>A1110501</td>
			<td>Used to lift osteoclast precursors</td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td>Used to stain differentiated osteoclasts</td>
		</tr>
	</tbody>
</table>

stimulation of notch signaling in mouse osteoclast precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

O objectivo deste método é a cultura e estimular a sinalização de Notch em precursores de osteoclastos. Quando adequadamente cultivadas, precursores de osteoclastos exibem uma morfologia fusiforme alongada essencialmente liso com citoplasma (Figura 1A). Cuidados devem ser tomados para evitar a activação imunológica dos precursores de osteoclastos. Após a activação, os precursores de espalhar e tornar-se achatado com espumoso citoplasma (Figura 1B).</str...

Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Estimulação da sinalização Notch no mouse osteoclastos Precursores

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

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