Recent studies utilizing small molecule antagonists indicate that the Parps Tankyrase 1 and 2 PARsylate and subsequently deactivate Axin1 and 2, components of the beta-catenin destruction complex (Fearon, 2009
; Huang et al., 2009
). In the presence of the tankyrase inhibitors, the Axins are stabilized and beta-catenin is degraded. Tnks1 and 2 activities are essential for beta-catenin signaling in cell culture and during zebrafish gut and tail fin regeneration. However, a general requirement of Tnks enzymes for canonical/beta-catenin dependent Wnt signaling in mammalian development has not been determined. Here, we show the broad expression of Tnks1 and 2 mRNA in multiple areas of active Wnt signaling during the embryonic period of the mouse. Further, we show, using several individual small molecule antagonists and ex vivo
organ culture, that tankyrase activity is required for normal embryonic development of the mouse kidney and lung. Finally, we demonstrate that the effects of the Tnks inhibitors on the kidney are the direct result of inhibition of the canonical/beta-catenin dependent Wnt pathway. These findings suggest that Tnks1 and 2 are core components of the Wnt transduction pathway during tissue development and maintenance.
The data presented here have uncovered a number of interesting findings related to Wnt signaling and kidney development. One such finding is that, in contrast to the situation in the regenerating zebrafish fin, tankyrase inhibition in the context of the developing mouse kidney is minimally reversible and the reversibility differs in distinct cell types. This limited reversibility of tankyrase inhibition in kidney seems to be attributable at least in part to the fact that reduced Wnt signaling in the context of the ureteric bud leads to premature differentiation of this tissue. These findings highlight differential roles for beta-catenin in diverse tissue types and perhaps are indicative of inherent differences between developing and regenerating tissues.
The distinct mode of action of the molecules utilized in this study should also allow researchers to tease apart canonical and non-canonical signaling events. Investigators can compare the blockade of all Wnt signaling (with IWP2), canonical signaling (with IWR1), or non-canonical signaling (with IWP2 supplemented with beta-catenin agonists). For instance, in this study, we found that at least in the context of tubule induction, the tankyrase inhibitors were always reversible by addition of LiCl while the porcupine inhibitors were minimally reversible. This finding may implicate a role for non-canonical Wnt signaling in maintaining the competence of the mesenchymal progenitor cells, a topic that is currently under investigation.
Relative to other commonly used Wnt inhibitors such as recombinant Dkk1, the small molecule inhibitors of tankyrase (as well as the inhibitors of porcupine) are extremely effective at inhibiting Wnt signaling in the context of organ culture (Iglesias et al., 2007
; Marose et al., 2008
). These molecules appear to completely block Wnt signaling at relatively low doses. In contrast, doses of Dkk1 up to 2ug/ml replaced every 12 hours had relatively mild effects on cultured kidneys (Data not shown). We predict this increased effectiveness will hold true for most, if not all, tissue types.
In addition to their potency, the IW molecules act rapidly (complete pathway repression is evident in less than 24 hours), appear to have no difficulty penetrating internal tissues and show little, if any, toxicity. This last point is supported by the fact that IW treated kidneys show defects very similar to Wnt and beta-catenin mutant kidneys and the phenotypes can be rescued (a the molecular level) by LiCl administration.
It is of note that the doses used in this study are in general 10-fold higher than those found to inhibit 50% of Wnt activity in cell culture. Interestingly, although XAV939 was found to be a much more potent inhibitor of tankyrase activity in vitro than IWR1, it was much less effective in the context of organ culture. The discrepancies in effectiveness of these compounds in vitro vs. in the context of organ culture may be the result of multiple factors. For instance, there may be differences in the half-lives of the compounds in the organ culture. Additionally the solubility of each factor will affect its effectiveness in the context of a complex, multi-cell layer tissue like the cultured kidney compared to cultured cells or a purely in vitro biochemical assay. The relatively high doses of these compounds required to completely block Wnt mediated events in organ culture may also indicate that very low levels of Wnt activity are sufficient to support normal development.
Finally, these compounds have additional advantages over current techniques of tissue specific gene ablation in that they do not depend on complicated, time consuming genetics or the efficiency of Cre mediated excision. For example, this study identified Pax2 as a potential beta-catenin target. This fact had not been revealed using traditional loss and gain of function studies. Further, with these compounds, the timing and degree of inhibition can be controlled and potentially reversed. Such studies can be undertaken in a relatively high throughput format utilizing small quantities of drug.
As TNKS1 and 2 mRNAs are present in multiple embryonic and adult tissues of humans, the Tnks inhibitors should be of wide use as biological and therapeutic reagents in multiple Wnt-related events and pathologies. In the kidney alone, beta-catenin misregulation has been implicated in Wilms’ tumors, nephronophthisis, kidney fibrosis and polycystic kidney disease (Benzing et al., 2007
; Guillen-Ahlers et al., 2008; He et al.,, 2009
; Koesters et al., 1999
, Lal et al., 2008
; Lin et al., 2003
; Major et al., 2007
; Qian et al., 2005
; Simons et al., 2005
; Surendran et al., 2002
). Unfortunately, IWR1 appears to be rapidly metabolized when administered to adult mice, limiting its usefulness as an in vivo
therapeutic reagent (Lu et al., 2009
). However, the organ culture system will facilitate medicinal chemistry studies focused on identifying more stable derivatives that retain their effectiveness.
In sum, we have utilized small molecule antagonists of Tankyrase 1 and 2 to reveal a broad general role for these factors in Wnt signaling during embryonic development. Tankyrase activity appears essential for all known Wnt pathway activity in the context of the developing kidney. The ability to chemically target these proteins will significantly enhance efforts to abrogate pathological and non-pathological Wnt signaling. Such antagonists will be invaluable as biological and therapeutic reagents.