Knockdown of TauT Expression Impairs Human Embryonic Kidney 293 Cell Development - Advances in Experimental Medicine and Biology - page 287

28.1 Introduction

Studies have shown that TauT is regulated by a variety of stresses, including tonic-
ity, oxidation, DNA damage, and dietary manipulation (Askwith et al. 2009 ; Han
et al. 2009 ; Matsell et al. 1997 ; Uchida et al. 1992 ) , suggesting that TauT is a stress
response gene. We and others (Han and Chesney 2010 ; Park et al. 2003 ) have dem-
onstrated that TauT is a target of c-Jun. c-Jun binds to two AP1 consensus sites in
the TauT promoter and upregulates TauT expression in renal cells. Mutation of AP1
sites blocked binding of c-Jun to the TauT promoter and further abolished the effect
of c-Jun on TauT regulation. Our previous studies have also shown that TauT is a
target gene of p53. Activation of p53 by a chemotherapeutic agent (cisplatin) sup-
presses TauT expression both in vitro and in vivo (Han et al. 2002 ; Han et al. 2009 ) .
We have also shown that the JNK signaling pathway is involved in the stress regula-
tion of TauT (Han and Chesney 2010 ). The JNK pathway is a major stress-signaling
pathway in cells that plays important roles in many cellular processes, including
development, apoptosis, and cell growth. In non-stressed cells, JNK targets the
ubiquitination and subsequent degradation of bound proteins such as c-Jun
(Han and Chesney 2010 ). In addition, JNK forms a complex with and degrades p53
(Fuchs et al. 1998 ). However, in stressed cells, JNK phosphorylates and activates
associated c-Jun and p53 proteins and enhances their transcriptional regulation of
stress-responsive genes (Buschmann et al. 2001 ; Derijard et al. 1994 ) . Based on
these results, we proposed a model for p53/c-Jun-mediated regulation of the TauT
gene in renal cells. In stressed cells, JNK phosphorylates and activates c-Jun and
p53 proteins and enhances their transcriptional regulation of TauT . Cisplatin-
induced activation of p53 decreases TauT promoter activity via the p53-inhibited
JNK-c-Jun pathway by competing with c-Jun for activation. Upon survival signal-
ing, c-Jun substitutes for p53 function and enhances TauT expression.
Models of TauT deficiency, which result in taurine deficiency, have been estab-
lished and studied during the past decade (Heller-Stilb et al. 2002 ; Ito et al. 2008 ) .
However, information about the consequences of TauT deficiency during cell devel-
opment and its possible effects on various signaling pathways is lacking. In the
present study, we used a plasmid-based shRNA, a sequence of TauT RNA that forms
a tight hairpin loop, to silence TauT gene expression via RNAi in 293 renal cells.
The TauT-deficient 293 cells were created and used to examine the effects of TauT
deficiency on renal cell development.

28.2 Materials and Methods

28.2.1 Cell Line and Culture Conditions

The 293 human embryonic kidney cells were obtained from the American Type
Culture Collection. The cells were grown in Dulbecco's modified Eagle's medium