Published On: Tue, Mar 26th, 2019

Jagged1 protein processing in the developing mammalian lens [RESEARCH ARTICLE]


The Notch signaling pathway is highly conserved among metazoans and functions in a variety of cellular activities, including cell fate determination. One developing tissue that relies on Notch signaling is the ocular lens. The vertebrate lens consists of anterior epithelial progenitor cells that terminally differentiate into organelle-free fiber cells. This is an attractive tissue for studying complex cell–cell signaling pathways, given its simple architecture and dispensability. Vertebrate lens induction initiates at E9.0 in the mouse when the surface ectoderm thickens into a lens placode (Charlton-Perkins et al., 2011; Piatigorsky, 1981). Lens placode formation relies on signals from the underlying optic vesicle, a neuroepithelium extension of the ventral forebrain that gives rise to both the neural retina and retinal pigment epithelium (Fuhrmann, 2010). At E10.5, the lens placode invaginates into a lens pit, which subsequently detaches from the surface ectoderm as a hollow lens vesicle, comprised entirely of progenitor cells (McAvoy et al., 1999). Then posterior progenitor cells, those closest to the developing retina, elongate as they differentiate into primary lens fiber cells. At the same time, anterior progenitor cells coalesce into the anterior epithelial layer (AEL). At E14.5, the lens switches its mode of development, when AEL cells begin to migrate to the equatorial transition zone, which constitutes a boundary between progenitors and differentiated fiber cells (reviewed in Mochizuki and Masai, 2014). Upon terminal differentiation, secondary fiber cells surround the primary fiber cells that comprise the central core of the lens. Previous studies demonstrated that Notch signaling regulates progenitor cell proliferation, secondary fiber cell differentiation and helps maintain the transition zone (Azimi et al., 2018; Jia et al., 2007; Le et al., 2009; Rowan et al., 2008; Saravanamuthu et al., 2009).

After ligand binding, Notch receptors undergo proteolysis, to release the Notch intracellular domain (N-ICD) polypeptide fragment (Bray, 2006; Schroeter et al., 1998). There are two broad classes of Notch ligands, Delta or Delta-like (Dll) and Serrate/Jagged (Jag), with mammalian genomes containing multiple paralogs for each type of ligand. Ligand truncations lacking either the extracellular or intracellular domain, fail to trigger Notch receptor activation, demonstrating that the entire ligand protein is necessary for functionality (Shimizu et al., 1999; Sun and Artavanis-Tsakonas, 1996). After receptor–ligand interaction, the Notch receptor is sequentially cleaved, with each event mediated by different enzymatic activities (reviewed in Kopan and Ilagan, 2009). First, ADAM proteases remove the Notch receptor extracellular domain, followed by γ-secretase protein complex cleavage within the transmembrane region, to release the N-ICD from the plasma membrane (Struhl and Greenwald, 1999). The γ-secretase complex is comprised of four proteins: Nicastrin, Aph-1, Pen2, and Presenilin (Psen), with Psen exhibiting the catalytic activity. Moreover, the γ-secretase complex cannot cleave full length Notch receptors, likely due to steric hindrance from the receptor extracellular domain. Thus, ADAM protease activity is a prerequisite for N-ICD generation (Bolduc et al., 2016; Struhl and Adachi, 2000). Once generated, the N-ICD fragment complexes with Rbpj and Mastermind proteins, which transcriptionally activate downstream genes within the nucleus (Wilson and Kovall, 2006).

In addition to Notch receptors, the γ-secretase complex cleaves at least 90 other substrates, including the amyloid precursor protein (APP), which accumulates abnormally in Alzheimer’s disease (Beel and Sanders, 2008; Haapasalo and Kovacs, 2011). Interestingly, previous in vitro studies showed that both Dll and Jag ligands can also be cleaved by γ-secretase (Ikeuchi and Sisodia, 2003; LaVoie and Selkoe, 2003; Six et al., 2003), and that overexpression of full length Dll or Jag in cultured cells facilitated the appearance of ligand-CTF isoforms, lacking extracellular ligand domains. This is consistent with the idea that ADAM proteases can cleave some of the Notch ligands. Furthermore, CTF isoforms accumulated after cells were treated with γ-secretase inhibitors, suggesting they are γ-secretase substrates. Although these data strongly suggest Notch pathway ligands as ADAM and/or γ-secretase substrates, the physiological relevance of ligand processing during development, homeostasis, or pathogenesis are essentially unknown. One recent in vivo study highlighted a role for the Jag1 intracellular domain (J1-ICD) in mouse adult cardiomyocytes (Metrich et al., 2015). Here J1-ICD overexpression reduced both Notch1 processing (N1-ICD levels), and the expression of downstream target genes Hes1 and Hey1/2. Moreover, increased J1-ICD expression correlated with reduced proliferation and premature cardiomyocyte differentiation. Therefore, Notch ligand processing may modulate signaling levels, via ligand turnover from the cell membrane and/or ligand intracellular domains could have intrinsic signaling activity.

Given that Notch receptor protein processing has been extensively studied and there remains sustained interest in pharmacologic interventions to block Notch dysregulation, deeper understanding of ligand protein regulation seems warranted. Here we show the presence of Jag1 protein isoforms during in vivo mouse lens development and test whether ADAM protease and γ-secretase activities regulate this ligand in vitro, using a human lens epithelial cell line. Moreover, mouse Jag1 colocalizes with Psen1 at three key ages of lens formation, consistent with the idea that γ-secretase can also cleave this ligand. We also describe multiple Jag1 protein isoforms in other developing mouse tissues, whose sizes correspond to full length Jag1, a Jag1-CTF intermediate, and the intracellular domain, J1-ICD. We found that human lens B3 cells endogenously express and process JAG1 protein, as well as transfected, epitope-tagged Jag1 isoforms that localized to the expected subcellular compartments. We also treated lens B3 cells with DAPT, which induced the accumulation of endogenous JAG1-CTF, the predicted γ-secretase substrate. Finally, J1-ICD overexpression in B3 cells stimulated an upregulation of endogenous JAG1 mRNA, suggesting that JAG1 protein isoforms can participate in Notch pathway feedback regulation.

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