PINCH1 is an adaptor/scaffolding protein that normally localizes at focal adhesion sites. In this study, we provide evidence demonstrating that PINCH1 also functions as a transcriptional regulator by interacting with nuclear WT1, a podocyte-specific transcription factor that plays a pivotal role in the establishment and maintenance of the unique differentiated features of podocytes in adult kidney. We show that PINCH1 is up-regulated in podocytes after stimulation with TGF-β1 and translocates into the nucleus, wherein it binds to WT1 and suppresses the WT1-mediated gene transcription. Our results uncover a novel function of PINCH1, in which it acts as a transcriptional regulator through controlling specific gene expression in podocytes.
Given the ability of PINCH1 to undergo nuclear translocation, it is conceivable that there are possible three subcellular (nuclear, cytoplasmic and focal adhesion-associated) pools of PINCH1 in podocytes. Each specific pool of PINCH1 may have different functions. While the focal adhesion-associated PINCH1 may play a critical role in modulating cell adhesion, cell shape and survival 
, we show here that nuclear PINCH1 is instrumental in regulating gene transcription via its interaction with WT1. Cellular stress/injury after TGF-β1 treatment accelerates the rate of its nuclear shuttling, resulting in an increased accumulation of PINCH1 in the nuclei, but it does not appear to significantly affect the focal adhesion-associated PINCH1 (). Nuclear accumulation of PINCH1 is unlikely a passive consequence of an increased overall level of its protein, since TGF-β1 also promotes nuclear translocation of PINCH1 after a short incubation (1–3 h) () when significant PINCH1 induction was not evident (). Not surprisingly, the putative NES/NLS motif in its C-terminus of PINCH1 is functionally important and obligatory for mediating its nuclear translocation, as deletion or site-directed mutations of this motif effectively prevents its nuclear shuttling () and its interaction with WT1 () in podocytes. Such a PINCH1 shuttling between cytoplasm and nucleus is also reported in Schwann cells after chronic constriction injury in adult rats 
. Consistently, several other LIM-containing proteins are found to be able to undergo nuclear shuttling 
. In this context, it is reasonable to conclude that PINCH1 is able to translocate into the nucleus in response to injury, thereby initiating new protein-protein interactions and participating in the control of gene transcription in diverse circumstances.
One of the novel findings in the present study is the identification of WT1 transcription factor as the binding partner for PINCH1 in the nuclei. Through defining the molecular details of PINCH1/WT1 interaction, we show that the LIM1 domain of PINCH1 mediates its interaction with WT1, whereas the C-terminal zinc-finger domains of WT1 are responsible for its binding to PINCH1 (). Because WT1, a key transcription factor that is exclusively expressed in glomerular podocytes in adult kidney, plays a critical role in establishing the unique features of podocytes by inducing specific gene expression, such a PINCH1/WT1 interaction likely has a detrimental consequence. Indeed, endogenous PINCH1/WT1 interaction actually occurs in podocytes after TGF-β1 stimulation (). Similarly, interaction between WT1 and the WT1-interacting protein (WTIP), another LIM-containing protein, is previously shown to lead to the suppression of WT1-mediated gene expression and podocyte dysfunction 
PINCH1 nuclear shuttling and subsequent interaction with WT1 could presumably influence the WT1-mediated gene expression in podocytes. In that regard, it is interesting to reveal that PINCH1 regulates the expression of podocalyxin, a well-characterized podocyte-specific protein that is transcriptionally controlled by WT1. Earlier in vivo
and in vitro
studies demonstrate that WT1 level and activity directly dictate podocalyxin expression in glomerular podocytes 
. Indeed, ectopic expression of WT1 in cultured podocytes induces podocalyxin mRNA and protein expression (). Given that PINCH1 binds to the zinc-finger domains of WT1, it is not unexpected that over-expression of PINCH1 abolishes WT1-mediated podocalyxin expression, while knockdown of PINCH1 induced podocalyxin expression (). Podocalyxin is a CD34-related, transmembrane, sialoglycoprotein that contains a highly charged cytoplasmic tail 
. It is connected to the cortical actin cytoskeleton via ezrin and Na+
-exchanger regulatory factor 2 (NHERF2) and plays an essential role in maintaining the foot process structure and filtration function. Disruption of podocalyxin/NHERF2/ezrin/actin interactions leads to pathologic conditions associated with changes in podocyte foot processes 
. Consistently, podocalyxin-deficient mice fail to form foot processes and slit diaphragms and die within 24 h after birth with anuric renal failure 
. Therefore, suppression of WT1-medated gene expression by PINCH1 could be a potential pathway leading to podocyte dysfunction.
In summary, we have shown that PINCH1 undergoes nuclear shuttling in podocytes after TGF-β1 stimulation. Nuclear PINCH1 via its LIM1 domain interacts with a new partner WT1. By interacting with the zinc finger domains of WT1, PINCH1 effectively blocks WT1-mediated gene transcription. These studies provide a proof of principal that PINCH1 can function as a transcriptional regulator by regulating specific gene expression.