The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.

We have previously demonstrated that transforming growth factor-beta(1) (TGF-beta(1)) rapidly activates the mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK signaling cascade, leading to the induction of type I collagen synthesis in mouse glomerular mesangial cells (Wang L, Ma R, Flavell RA, Choi ME. J Biol Chem 277: 47257-47262, 2002). In the present study, we investigated the functional role of upstream TGF-beta-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) in the TGF-beta(1) signaling cascade. Rapid activation of endogenous TAK1 activity by TGF-beta(1) was observed in mouse mesangial cells. Transient overexpression of TAK1 with TAB1 enhanced the activation of MKK3 and p38 MAPK with or without TGF-beta(1) stimulation, whereas a dominant-negative mutant of TAK1 (TAK1DN) suppressed TGF-beta(1)-induced activation of MKK3 and p38 MAPK. Moreover, constitutive expression of TAK1DN reduced steady-state protein levels of MKK3 and p38 MAPK as well as MKK3 phosphorylation. Increased p38alpha MAPK activity by ectopic expression of either TAB1 or wild-type p38alpha MAPK resulted in enhanced TGF-beta(1)-induced type I collagen expression. In contrast, constitutive expression of TAK1DN inhibited collagen induction. Taken together, our data indicate that TAK1 and TAB1 play a pivotal role as upstream signal transducers activating the MKK3-p38 MAPK signaling cascade that leads to the induction of type I collagen expression by TGF-beta(1). In addition, our findings also suggest that TAK1 has a novel function in regulation of the steady-state protein levels of MKK3 and p38 MAPK.

Transforming growth factor-␤1 (TGF-␤1) is a multifunctional cytokine that signals through the interaction of type I (T␤RI) and type II (T␤RII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-␤1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-␤1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with T␤RI. TGF-␤1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of T␤RI is crucial for its interaction with TAK1. Both T␤RI-mediated TAK1 phosphorylation and TGF-␤1-induced TAK1 phosphorylation do not require kinase activity of T␤RI. Moreover, T␤RI-mediated TAK1 phosphorylation correlates with the degree of its association with T␤RI and requires kinase activity of TAK1. TAB1 does not interact with TGF-␤ receptors, but TAB1 is indispensable for TGF-␤1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with T␤RI and TGF-␤1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-␤1-induced interaction of T␤RI and T␤RII triggers dissociation of TAK1 from T␤RI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of T␤RI.