Rabconnectin-3 is a functional regulator of mammalian Notch signaling

Abstract

The Notch signaling pathway is important for cell fate decisions in embryonic development and adult life. Defining the functional importance of the Notch pathway in these contexts requires the elucidation of essential signal transduction components that have not been fully characterized. Here, we show that Rabconnectin-3B is required for the Notch pathway in mammalian cells. siRNA-mediated silencing of Rabconnectin-3B in mammalian cells attenuated Notch signaling and disrupted the activation and nuclear accumulation of the Notch target Hes1. Rabconnectin-3B knockdown also disrupted V-ATPase activity in mammalian cells, consistent with previous observations in Drosophila. Pharmacological inhibition of the V-ATPase complex significantly reduced Notch signaling in mammalian cells. Finally, Rabconnectin-3B knockdown phenocopied functional disruption of Notch signaling during osteoclast differentiation. Collectively, these findings define an important role for Rabconnectin-3 and V-ATPase activity in the Notch signaling pathway in mammalian cells.

FIGURE 1.

siRNA-mediated knockdown of Rbcn-3 disrupts Notch signaling in HaCaT cells. A, γ-secretase inhibitor treatment disrupts Notch signaling in the HaCaT cell line as shown by Western blot analysis of Notch1 intracellular domain (Notch1^ICD) protein levels. B, qRT-PCR mRNA expression levels of Hey1 in HaCaT cells treated with different durations of GSI-IX. C, qRT-PCR mRNA levels of Rbcn-3 in HaCaT cells treated with a control or three Rbcn-3 siRNAs. D, Notch reporter levels in HaCaT cells treated with a control or three Rbcn-3 siRNAs by luciferase assay. Notch reporter firefly luciferase counts were normalized to Renilla luciferase counts. E, qRT-PCR mRNA levels of several Notch target genes in HaCaT cells treated with a control or three Rbcn-3 siRNAs. Data represent average ± S.D. *, p < 0.05; **, p < 0.01 by Student's t test.

FIGURE 2.

siRNA-mediated knockdown of Rbcn-3 reduces expression levels and disrupts localization of the Notch target Hes1 in HaCaT skin and MCF-7 breast cancer cells. A, localization of Hes1 and pSmad2 in HaCaT cells treated with a control or three Rbcn-3 siRNAs by immunofluorescence. Scale bar, 100 μm. B, localization of Hes1 in MCF-7 breast cancer cells treated with a control or three Rbcn-3 siRNAs by immunofluorescence. Scale bar, 100 μm.

FIGURE 3.

Rbcn-3 is required for ATPase activity in HaCaT keratinocytes and MCF-7 breast cancer cells. A, LysoTracker staining of acidic intracellular compartments in HaCaT cells treated with a control or three Rbcn-3 siRNAs by immunofluorescence. Scale bar, 100 μm. B, Lysotracker staining of acidic intracellular compartments in MCF-7 cells treated with a control or three Rbcn-3 siRNAs by immunofluorescence. Scale bar, 100 μm.

FIGURE 4.

Bafilomycin A1 treatment disrupts Notch signaling in mammalian cells. A, LysoTracker staining of acidic intracellular compartments in HaCaT cells treated with DMSO, 1 μm GSI, or 0.5 μm bafilomycin A1. Scale bar, 100 μm. B, Notch reporter activity in HaCaT cells treated with the indicated concentrations of bafilomycin A1 and GSI IX. C, Notch reporter activity in Jagged1-overexpressing tumor cell coculture system treated with the indicated concentrations of bafilomycin A1 and GSI. Notch reporter firefly luciferase counts were normalized to Renilla luciferase counts. B and C, data represent average ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student's t test. D, localization of Hes1 in MCF-7 breast cancer cells treated with DMSO, bafilomycin A1, or GSI IX by immunofluorescence. Scale bar, 30 μm.

FIGURE 5.

Loss of Rbcn-3 phenocopies defective Notch signaling in osteoclast differentiation. A, quantification of TRAP⁺ multinucleated MOCP5 osteoclasts treated with a control or three Rbpj siRNAs (left) or three Rbcn-3 siRNAs (right). B, quantification of TRAP⁺ multinucleated Raw 264.7 osteoclasts treated with a control or three Rbpj siRNAs (left) or three Rbcn-3 siRNAs (right). A and B, data represent average ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student's t test. C, representative images of TRAP⁺ Raw 264.7 osteoclasts from the distinct experimental groups. Scale bar, 200 μm. D, left panel, localization of Hes1 in MOCP5 pre-osteoclasts treated with a control or three Rbcn-3 siRNAs by immunofluorescence. Right panel, representative images of TRAP⁺ MOCP5 osteoclasts treated with a control or three Rbcn-3 siRNAs, bottom panel. Scale bar, 200 μm.