TAZ/Wnt-β-catenin/c- axis regulates cystogenesis in polycystic kidney disease

Eun Ji Leea,1, Eunjeong Seob,1, Jin Won Kimb, Sun Ah Namb, Jong Young Leeb, Jaehee Juna, Sumin Oha, Minah Parka, Eek-hoon Jhoc, Kyung Hyun Yooa, Jong Hoon Parka,2, and Yong Kyun Kimb,d,2

aDepartment of Biological Science, Sookmyung Women’s University, 04310 Seoul, Republic of Korea; bCell Death Disease Research Center, College of Medicine, The Catholic University of Korea, 06591 Seoul, Korea; cDepartment of Life Science, University of Seoul, 02504 Seoul, Republic of Korea; and dDepartment of Internal Medicine, College of Medicine, The Catholic University of Korea, St. Vincent’s Hospital, 16247 Suwon, Republic of Korea

Edited by Janet Rossant, The Gairdner Foundation, Toronto, ON, Canada, and approved September 28, 2020 (received for review May 11, 2020) Autosomal-dominant polycystic kidney disease (ADPKD) is the pathway, increased β-catenin activity in the kidneys (12). Fur- most common genetic renal disease, primarily caused by germline thermore, Wnt/β-catenin signaling or its target such as mutation of PKD1 or PKD2, leading to end-stage renal disease. The c-Myc may regulate cystogenesis in the mouse kidney (11, 13, 14). Hippo signaling pathway regulates growth and cell prolif- Therefore, we focused on deciphering the role of TAZ, a Hippo eration. Herein, we demonstrate the regulatory mechanism of cys- effector, in regulating cystogenesis in ADPKD. togenesis in ADPKD by transcriptional coactivator with PDZ- Herein, we elucidate a mechanism by which TAZ promotes binding motif (TAZ), a Hippo signaling effector. TAZ was highly the activation of Wnt/β-catenin signaling in the kidney of Pkd1- expressed around the renal cyst-lining epithelial cells of Pkd1- deficient mice and show that it increases c-Myc transcript levels. deficient mice. Loss of Taz in Pkd1-deficient mice reduced cyst for- Basal YAP1/TAZ expression levels were high around the cyst- mation. In wild type, TAZ interacted with PKD1, which inactivated lined cells in the kidneys of Pkd1-deficient mice and patients with β-catenin. In contrast, in PKD1-deficient cells, TAZ interacted with ADPKD and were associated with high c-MYC and β-catenin AXIN1, thus increasing β-catenin activity. Interaction of TAZ with expression levels. The loss of TAZ in Pkd1-deficient mice AXIN1 in PKD1-deficient cells resulted in nuclear accumulation of resulted in low levels of the c-MYC and was observed to TAZ together with β-catenin, which up-regulated c-MYC expres- delay the progression of PKD. In vitro studies revealed that sion. Our findings suggest that the PKD1–TAZ–Wnt–β-catenin– PKD1 mostly interacted with TAZ in wild-type cells, but its c-MYC signaling axis plays a critical role in cystogenesis and might absence allowed TAZ to strongly interact with AXIN1, thereby MEDICAL SCIENCES be a potential therapeutic target against ADPKD. resulting in a weak interaction between β-catenin and AXIN1. We further observed that c-MYC expression, which causes cys- polycystic kidney | TAZ | c-myc togenesis in mouse kidney, was directly regulated by TAZ and β-catenin in PKD1-deficient cells. Overall, our results show that utosomal-dominant polycystic kidney disease (ADPKD) is the TAZ–β-catenin–c-MYC axis is responsible for renal cysto- Athe most common inherited kidney disease, caused by ge- genesis in Pkd1-deficient mice. Based on these findings, we netic mutations in PKD1 or PKD2, which leads to end-stage renal disease. Polycystins, the transmembrane encoded by Significance PKD1 or 2, are nonselective cation channels transporting calcium ions into the cells. Disruption of polycystic kidney disease (PKD) Autosomal-dominant polycystic kidney disease (ADPKD) is the genes impairs intracellular calcium homeostasis and results in most common genetic renal disease, primarily caused by the development of numerous fluid-filled cysts from abnormally germline mutation of PKD1 or PKD2, leading to end-stage proliferating renal tubular cells. It is also accompanied by in- renal disease. There are few cures for ADPKD, although terstitial inflammation and fibrosis around the cyst-lining cells, many researchers are trying to find a cure. The Hippo signaling ultimately reaching end-stage renal disease (ESRD) (1, 2). pathway regulates organ growth and . Tran- The Hippo signaling cascades are essential to control organ scriptional coactivator with PDZ-binding motif (TAZ) is a Hippo size, differentiation, and tissue regeneration. These are highly signaling effector. In this study, we demonstrated that the coordinated processes in which more than 30 core proteins are PKD1–TAZ–Wnt–β-catenin–c-MYC signaling axis plays a critical involved in responding to the mechanical stimuli from the cel- role in cystogenesis. Endo IWR1 treatment, which inhibited lular microenvironment. Activated Hippo cascades, in- β-catenin activity via AXIN stabilization, reduced cyst growth in cluding sterile 20-like kinase 1/2 (MST1/2) and large tumor an ADPKD model. Our findings provide a potential therapeutic suppressor 1/2 (LATS1/2), lead to the phosphorylation of their target against ADPKD and would be important for clinical downstream effectors, YAP1/TAZ, followed either by their cy- translation. tosolic retention or degradation, thus preventing their nuclear localization (3–5). Inactivation of the Hippo pathway increases Author contributions: E.J.L., E.S., J.J., E.J., J.H.P., and Y.K.K. designed research; E.J.L., E.S., the nuclear localization of YAP1 and TAZ, which interact with J.W.K., S.A.N., J.Y.L., J.J., and M.P. performed research; E.J.L., E.S., E.J., J.H.P., and Y.K.K. contributed new reagents/analytic tools; E.J.L., E.S., J.W.K., S.A.N., J.Y.L., J.J., S.O., M.P., the TEAD family transcription factors and drive the expression E.J., K.H.Y., J.H.P., and Y.K.K. analyzed data; and E.J.L., E.S., J.H.P., and Y.K.K. wrote of target genes, such as CTGF, CYR61, and c-MYC (4, 6). the paper. Recently, the role of Hippo signaling pathway has emerged in The authors declare no competing interest. the formation of cysts in ADPKD (7, 8). A previous study has This article is a PNAS Direct Submission. c-MYC reported that YAP1 and its transcriptional target, , me- This open access article is distributed under Creative Commons Attribution-NonCommercial- diate cystic kidney pathogenesis in Pkd1-deficient mice, and a NoDerivatives License 4.0 (CC BY-NC-ND). – – RhoA YAP c-MYC axis was suggested to be involved in the 1E.J.L. and E.S. contributed equally to this work. – pathogenesis of ADPKD (9 11). 2To whom correspondence may be addressed. Email: [email protected] or However, the role of Hippo signaling pathway in cystogenesis [email protected]. in ADPKD remains unclear. TAZ may regulate the Wnt/β-cat- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ enin signaling. We previously reported that intrinsic activation of doi:10.1073/pnas.2009334117/-/DCSupplemental. TAZ by genetic deletion of WW45, a component of the Hippo First published October 29, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2009334117 PNAS | November 17, 2020 | vol. 117 | no. 46 | 29001–29012 Downloaded by guest on September 24, 2021 suggest that the TAZ–β-catenin–c-MYC axis is a potential ther- to those in Pkd1-deleted mice, indicating that reduced TAZ apeutic target for ADPKD. expression enhanced renal function (Fig. 3B). Next, we examined whether the increased expression of active β-catenin and c-MYC Results in the kidney of Pkd1-null mice was modulated by the reduction YAP1/TAZ and c-MYC Are Increased in the Kidney of Pkd1-Targeted in TAZ levels. As a result, TAZ mutation in Pkd1-deleted mice Mice and Patients with ADPKD. We used renal collecting duct- reduced the levels of active β-catenin and c-MYC in the kidney specific Pkd1-knockout mice as previously described (15). To (Fig. 3C). Immunofluorescent analyses also revealed that the determine whether the expression of TAZ correlated with those increased signals were significantly reduced in Pkd1/Taz double- of β-catenin and c-MYC in Pkd1-null kidney, we first assessed knockout kidneys (Fig. 3 D–F). In addition, cell proliferation, protein levels of TAZ and c-MYC in the kidney of Pkd1-deleted which was hyperactivated around the cysts derived by Pkd1 de- mice and found that their levels, along with the level of active letion, was inhibited in double-knockout kidneys (Fig. 3G). β-catenin protein, were highly up-regulated (Fig. 1A). The Consistently, a strong increase in fibrosis score in Pkd1-null mRNA levels of c-Myc, a known target of β-catenin or YAP1/ kidneys, which was indicated either by Masson’s trichrome TAZ, were enhanced in the kidney of Pkd1-null mice (Fig. 1B). staining or immunohistochemical staining for collagen IV, was To ensure the collecting duct-specific deletion of Pkd1 that is restored in Pkd/Taz double-knockout ones (Fig. 3H). Collec- followed by the development of renal cysts, we costained col- tively, these results suggested that reduction in the TAZ ex- lecting duct-specific marker (DBA) with target proteins. We pression alleviated the PKD and was accompanied by have confirmed that all of the cyst-lined cells were stained with decreased expression of active β-catenin and c-MYC. DBA, and, furthermore, accumulation of TAZ and c-MYC was increased, in the Pkd1-deleted kidneys (Fig. 1 C–F). In addition, In Vitro Cystogenesis Is Stimulated by the Increase in TAZ Levels, and β-catenin was activated in both cyst-lined epithelia and periph- Wnt Inhibition Attenuates Its Effect. TAZ is one of the upstream eral cells around the cyst linings (Fig. 1 G and H). In line with regulators of c-MYC expression, both implicated in renal cys- these experimental data, we examined TAZ, active β-catenin, togenesis (2, 6). Since TAZ and c-MYC levels were increased in total β-catenin, and c-MYC expression in normal and the kidney of Pkd1-null mice, we examined whether an increase ADPKD patient kidney. The indicated proteins were highly in TAZ up-regulated the expression of c-MYC in Pkd1-silenced expressed in cystic lining cells of ADPKD patient kidney com- cells. Consistent with previous observations, Pkd1 silencing in- pared to normal human kidney tissues (Fig. 1I). These results creased TAZ and c-MYC levels in IMCD cells (Fig. 4 A and B). indicated that the overexpression of TAZ was positively corre- Next, we evaluated the protein levels of c-MYC in cells treated lated to that of β-catenin and c-MYC in the kidneys of both with a cell-permeable imidazole-[4,5-b] pyridine derivative, a Pkd1-deleted mice and patients with ADPKD. TAZ nuclear-promoting chemical that enhances the nuclear lo- calization of TAZ. The treatment had enhanced the amount of RNA-Seq Analysis Showed Significant Increase of Yap/Taz Target TAZ protein in a concentration-dependent manner with a con- Expression in Pkd1-Deleted Kidneys. For more in-depth analysis, comitant increase in c-MYC expression (Fig. 4C). To further alterations in the target genes expression were verified on an investigate whether changes in either the genetic expression or mRNA level based on RNA-seq data, which had been previously the activity of TAZ affected cyst growth in vitro, we performed accomplished using kidney tissues from the same mice model 3D culture of IMCD cells. The cells were pretreated with siRNA (15). We first screened changes in Yap, Taz, and β-catenin levels targeting Pkd1 or Taz for 24 h and embedded in Matrigel (in a and confirmed that expression of those genes insignificantly 1:1 ratio with the culture medium). Subsequently, DMEM F/12 changed in Pkd1-deleted kidneys (Fig. 2A). Meanwhile, different medium with 5 μM forskolin was added, and cysts were observed expression patterns were observed in Yap/Taz target genes, on the fifth day of seeding. Forskolin increases the intracellular which have been listed from public sources comprising 354 cAMP level, and thereby provides PKD- mimicking conditions genes. We found that an enrichment score (ES) of the rank- and stimulates cyst growth. We observed the gradual develop- ordered Yap/Taz target genes showed highly increased patterns ment of cysts 2 to 3 d following forskolin treatment, while Pkd1 by GSEA analysis (Fig. 2B). Of the Yap/Taz target genes, 129 silencing led to an increased cystic area. Cysts developed from genes were defined as leading-edge subset genes, which had cells silenced with siRNAs targeting Pkd1 and Taz were smaller significantly higher expression levels in Pkd1-deleted cystic kid- (Fig. 4D). We also observed the effect of TAZ overexpression on neys, and the top 20 genes differentially expressed with log2 fold the growth of in vitro cysts. Forced expression of TAZ changes of greater than 0.795 were indicated (Fig. 2 C and D). accelerated cystogenesis of Pkd1-silenced cells, as indicated by Among them, high-rank genes including c-Myc were validated the increase in the cystic area compared to the cells transfected using quantitative real-time PCR (qRT-PCR), and the expres- with the Pkd1-targeting siRNA alone (Fig. 4E). Next, we ex- sion level of those genes was indeed increased (Fig. 2E). These amined whether treatment with a drug regulating TAZ activity results further suggested that Yap/Taz activation might be pos- (TAZ modulator) affected cyst growth. Our results showed that itively implicated in the growth of renal cysts stimulated by the in vitro cysts were significantly increased by drug treatment Pkd1 deletion. and were associated with enhanced c-MYC levels (Fig. 4F). We further investigated whether the inhibition of β-catenin activity Deletion of Taz Inhibits Cyst Growth with Enhanced Renal Function in could attenuate the cyst-stimulating effect of the TAZ modula- the Kidney of Pkd1-Targeted Mice. To elucidate the role of en- tor. Endo IWR1, an AXIN stabilizer, was used to down-regulate hanced TAZ levels in renal cyst growth in the kidneys of Pkd1- β-catenin activity (16). Treatment with Endo IWR1 successfully targeted mice, we generated collecting duct-specific double inhibited active β-catenin, followed by a decrease in c-MYC level f/f f/f Pkd1-/Taz-knockout mice (Pkd1 ;Taz ;HoxB7Cre). These mice (Fig. 4G). A negative control Exo IWR1, conversely, did not were euthanized on postnatal day 13. Pkd1-deficient mice with affect either. Observations in 3D culture showed that in vitro Taz deletion showed highly reduced cyst development. The cystic cysts stimulated by TAZ modulator were significantly reduced by area was quantified to indicate the changes in its size distribu- Endo IWR treatment (Fig. 4H). We additionally verified the tion, and it indeed revealed that the number of large cysts was effect of mTOR inhibitor, which was previously reported to re- significantly decreased in Pkd1/Taz double-knockout kidneys duce cyst growth in multiple preclinical models, on TAZ/β-catenin/ (Fig. 3A). In addition, the kidneys to total body weight (2KW/ c-MYC pathways. We first investigated whether rapamycin af- TBW) ratio, as well as blood urea nitrogen (BUN) level, in Pkd1/ fects the TAZ/Wnt/β-catenin/c-MYC axis and found it to be Taz double-knockout mice were significantly lowered compared significantly inhibited by drug treatment. In addition, treatment

29002 | www.pnas.org/cgi/doi/10.1073/pnas.2009334117 Lee et al. Downloaded by guest on September 24, 2021 MEDICAL SCIENCES

Fig. 1. High expression of TAZ, c-MYC, and active β-catenin in the kidneys of Pkd1-deleted mice and patients with ADPKD. (A) Screening of the basal levels of YAP/TAZ, c-MYC, and active β-catenin in the kidneys of Pkd1-deleted mice at postnatal day 13 (p13). (B) Increased c-Myc levels in mRNA samples from Pkd1- deleted kidneys. (C–H) Immunofluorescent (IF) staining of DBA, TAZ, c-MYC, and active β-catenin (ABC) around the cyst-lining cells in the kidneys of Pkd1- deleted mice. Fluorescent signals were quantified by the green histogram analyses using ImageJ. (I) Immunohistochemistry of normal and ADPKD human kidney. Immunohistochemical (IHC) staining of TAZ, β-catenin, active β-catenin (ABC), and c-MYC. TAZ, β-catenin, active β-catenin (ABC), and c-MYC were substantially increased around the cyst-lining cells of ADPKD patients compared to the normal kidney. (Scale bar, 100 μm.) Two wild-type and four Pkd1-null mouse kidneys were used to test basal expression levels of TAZ, c-MYC, and active β-catenin. Statistical analysis was performed using two-tailed t tests. A value of P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

Lee et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 29003 Downloaded by guest on September 24, 2021 ABCD 25000 Enrichment plot: YAP/TAZ target genes Pkd1f/f;HoxB7cre Pkd1f/f Top 20 significant genes Pkd1f/f 0.35 #1 #2 #3 #1 #2 #3 f/f ES : 0.3476 Ankrd1 Pkd1 ;HoxB7cre 0.30 FDR q-value < 0.0001 Fgf5 20000 0.25 Serpine1 0.20 Foxf2 0.15 Gadd45b Cyr61 0.10 15000 Ctgf 0.05 Tnfrsf12a Enrichment score (ES) 0.00 Fam46b Bcat1 F3 10000 Igfbp3

129 cutting edge Fst 10 positively correlated Anxa3 5 Fjx1 5000 0 Dkk1 Zero crosso att 82588 Myc -5

Expresssion levels (Read count) Gins1 -10 negatively correlated Tubb6 0 5,000 10,000 15,000 20,000 Arntl2

0 Ranked list metric (PreRanked) 01234 5 Enrichment profile Hits Ranking metric scores Yap1 Taz Ctnnb1 min log2 (Fold change)

E 5 25 30 *** 6 4 8 ** *** *** *** ** 4 20 3 6 20 4 3 15 2 4 mRNA level

2 10 mRNA level mRNA level 10 2 1 2 1 5 Ctgf c-Myc mRNA level Cyr61 Ankrd1 mRNA level Serpine1 Cadd45d mRNA level

0 0 0 0 0 0 Pkd1f/f Pkd1f/f;HoxB7cre Pkd1f/f Pkd1f/f;HoxB7cre Pkd1f/f Pkd1f/f;HoxB7cre Pkd1f/f Pkd1f/f;HoxB7cre Pkd1f/f Pkd1f/f;HoxB7cre Pkd1f/f Pkd1f/f;HoxB7cre

Fig. 2. analyses of TAZ and its target genes in Pkd1-deleted kidneys based on RNA-seq data. (A) Changes of Yap, Taz, and β-catenin levels in Pkd1-deleted kidneys compared to control. (B) GSEA analysis of 354 Yap/Taz target genes between Pkd1-deleted kidneys and controls. (C) Heat map showing 129 leading-edge subset genes, which are significantly increased in Pkd1-deleted kidneys. These are results from n = 3 individual samples per group. (D) log2 fold change values in top 20 increased genes in Pkd1-deleted kidneys compared to normal. (E) Validation of mRNA expression levels of high-ranking genes including c-Myc. Statistics were analyzed by t tests, and P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

with rapamycin combined with the Wnt inhibitor showed sig- β-catenin, which was enhanced to the level comparable with nificant decrease of in vitro cyst growth compared to that ob- TAZ-expressing cells upon coexpression of PKD1 and TAZ served in the rapamycin-treated group (SI Appendix, Fig. S1). (Fig. 4D). Collectively, these results suggest that TAZ-dependent Therefore, we believe that TAZ and its downstream signaling PKD1 expression negatively regulates the activation of β-catenin. pathways mediated by c-MYC negatively impact cyst develop- β-Catenin is degraded by an AXIN1-containing destruction ment and thus PKD progression. complex. Therefore, we examined whether AXIN1 participated in the PKD1-mediated regulation of β-catenin activation. Our β Regulation of -Catenin Activation by PKD1 through TAZ and AXIN1. results showed that AXIN1 depletion restored the levels of ac- Pkd1 We observed that the kidneys of -deleted mice showed an tive β-catenin in PKD1-expressing cells (Fig. 5E). Further, increase in the levels of TAZ along with high levels of active AXIN1 depletion also obstructed the reduction of the tran- β-catenin and c-MYC proteins (Fig. 2 A and B). TAZ is known to β c-MYC scriptional activity of -catenin in PKD1-expressing cells induce the expression of mRNA overlap with the target (Fig. 5F). These data indicate that AXIN1 may be a downstream gene of Wnt/β-catenin signaling (6). Next, we determined the mediator of PKD1 in regulating the β-catenin activity in PKD1- TAZ–β-catenin–c-MYC downstream signaling of PKD1 in de- depleted cells. We further determined the epistatic relationship tail. For this, we first investigated whether PKD1 or TAZ de- between TAZ and AXIN1 underlying the PKD1 function in the pletion or a codepletion affected the expression of active β β-catenin. PKD1 depletion induced a slight increase in TAZ regulation of the transcriptional activity of -catenin. The ex- levels and significantly increased the levels of active β-catenin. pression of TAZ shRNA reduced the transcriptional activity of β-catenin, which was significantly increased by AXIN1 silencing Further, this increase was reduced to a level comparable to that G in control cells upon transfection of PKD1 siRNAs in TAZ (Fig. 5 ). Furthermore, AXIN1 expression led to a strong re- β shRNA-expressing cells (Fig. 5A). Conversely, the feeble ex- duction in -catenin activation induced by TAZ expression G pression of active β-catenin in control cells was abrogated by the (Fig. 5 ). These results suggest that TAZ acts upstream of β overexpressed Flag-PKD1, whereas the expression of HA-TAZ AXIN1 to regulate the transcriptional activation of -catenin. To markedly increased the active β-catenin levels in a dose- confirm whether TAZ-dependent PKD1 expression affected the dependent manner, irrespective of the forced expression of expression of the target genes of β-catenin, we examined the Flag-PKD1 (Fig. 5B). In line with these results, we further in- effects of PKD1 silencing alone or of PKD1 and TAZ silencing vestigated whether the transcriptional activity of β-catenin was on the expression of AXIN, c-MYC, and CCND2 mRNAs; the regulated under the same conditions as presented in Fig. 5A. The expression levels of these genes were elevated in PKD1-depleted depletion of PKD1 led to a strong induction of the transcrip- cells but not in TAZ-deficient cells expressing PKD1 (Fig. 5H). tional activity of β-catenin, which was reduced by the knockdown These data indicate that the regulation of β-catenin–mediated of TAZ in PKD1-depleted cells (Fig. 5C). In contrast, PKD1 transcriptional activation of AXIN2, c-MYC, and CCND2 by expression slightly decreased the transcriptional activity of PKD1 depends on TAZ.

29004 | www.pnas.org/cgi/doi/10.1073/pnas.2009334117 Lee et al. Downloaded by guest on September 24, 2021 Fig. 3. Alleviation of PKD with enhanced renal function in the kidneys of Taz-deleted Pkd1-deficient mice. (A) Hematoxylin and eosin (H&E)- stained sections of wild-type, Taz-null, Pkd1-null, or Pkd1/Taz double-null kid- neys. Renal cystic area either of Pkd1- null or Pkd1/Taz double-null kidneys was quantified by ImageJ, and graph shows the changes in size distributions. One wild-type, 2 Taz-null, 11 Pkd1-null, and 6 Pkd1/Taz double-null kidneys were used for quantification. At least 70 cysts for each individual kidney were sized and quantified. (B) Ratio of two kidneys’ weight to total body weight and blood urea nitrogen level in mice at postnatal day 13 (p13). The number of mice used for comparing 2KW/TBW ratio and BUN level is indicated as dots for each individual. (C) Western blot analy- sis using whole kidney lysates from each groups of mice at p13 to test the changes in c-MYC and β-catenin levels MEDICAL SCIENCES caused by TAZ deficiency. (D–F) Immu- nofluorescent staining of TAZ, c-MYC, or active β-catenin and quantitative analy- ses using ImageJ. (G) Changes of cell proliferation tested by Ki-67 staining. Fluorescent signals were quantified as described above. One wild-type, two Taz-null, three Pkd1-null, and four Pkd1/ Taz double-null kidneys were used for immunofluorescent staining of target proteins. The number of images used for statistics are indicated as dots in the graph. Statistical analyses for A to G were performed using two-tailed t tests. A value of P < 0.05 was considered sta- tistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). (H) Masson’s tri- chrome histology and immunostaining for collagen IV of genotype kidneys. Pkd1-knockout kidney revealed an in- crease of fibrosis, which was reduced in Pkd1/Taz double-knockout kidney. All results are representative of at least three mice per genotype in two inde- pendent experiments. Each bar repre- sents the mean ± SEM (*P < 0.05 compared with the wild-type mice; #P < 0.05 compared with the Pkd f/f;HoxB7 cre mice). (Scale bar, 100 μm.)

A Strong Interaction of AXIN1 with TAZ in the Absence of PKD1 with AXIN1 in the absence of PKD1 (Fig. 6A). These observa- Causes the Activation of β-Catenin. β-Catenin activation is regu- tions suggest that TAZ interacts more strongly with PKD1 than lated by PKD1, which in turn depends on TAZ and AXIN1 with AXIN1, whereas the absence of PKD1 triggers the inter- (Fig. 5). TAZ is known to associate with the β-catenin destruc- action of TAZ with AXIN1. We assumed that the interaction of tion complex (17) or to interact with PKD1 (18). We therefore TAZ with AXIN1 in the absence of PKD1 influences the asso- tested the possible interaction of TAZ with AXIN1 and PKD1 ciation of AXIN1 with β-catenin. To confirm this, Myc-AXIN1 using PKD1-silenced cells transfected with HA-TAZ. Our results was pulled down with the Myc antibody from the lysates of Myc- showed that PKD1 depletion did not change the amount of total AXIN1–expressing cells transfected with si-PKD1 and si-control. HA-TAZ, which was thought to be saturated in cells. HA-TAZ Immunoblotting of the Myc-AXIN1 immunoprecipitated with coprecipitated with endogenous PKD1, whereas it interacted anti–β-catenin antibody revealed that β-catenin coprecipitated

Lee et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 29005 Downloaded by guest on September 24, 2021 Fig. 4. Changes in c-MYC levels by controlling of TAZ, followed by in vitro cyst growth. (A and B) Screening of TAZ and c-MYC expression levels in Pkd1- silenced mouse inner medullary collecting duct (IMCD) cells. (C) Effect of TAZ modulator, which enhances TAZ activities via translocation into the nucleus. Cells were treated with the drugs at different concentrations from 1 to 10 μM. The level of TAZ was increased at both low and high concentrations and was followed by increased expression of c-MYC. (D–F) The effect of controlling TAZ levels on in vitro cystogenesis, either by genetic regulation or treatment of TAZ modulator. siRNA successfully targeted TAZ, and, conversely, the TAZ-expressing vector overexpressed the TAZ protein with consequent increaseinthe expression of c-MYC. Cells pretransfected for 24 h were embedded in Matrigel, and culture medium with 10 μM forskolin was added on the next day. The medium was changed daily. In vitro cysts were observed at 5 d after embedding. Taz silencing decreased the in vitro enlargement of cysts, whereas over- expression or enhancement of TAZ activity stimulated in vitro cystogenesis. (G) The changes in c-MYC level by Wnt inhibition. Either Exo IWR1 (a negative control of Endo IWR1) or Endo IWR1 (the AXIN stabilizer) was administered at 10 μM for 24 h. (H) The effect of Wnt inhibitors on in vitro cysts stimulated by TAZ modulator. Pretransfected cells were embedded in Matrigel, three-dimensionally cultured as described above, and finally observed at 5 d after seeding. All data were obtained from a minimum of three independent experiments. Statistical analysis was performed using two-tailed t tests. A value of P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

29006 | www.pnas.org/cgi/doi/10.1073/pnas.2009334117 Lee et al. Downloaded by guest on September 24, 2021 Fig. 5. Regulation of β-catenin activation by PKD1 through TAZ and AXIN1. (A) Inhibition of increased active β-catenin in PKD1-silenced cells expressing shTAZ viral vectors. Inner medullary collecting duct (IMCD) cells were transduced with retroviral vectors expressing shTAZ or with the indicated siRNAs, and Western analysis was performed. (B) Increase in the levels of active β-catenin in a dose-dependent manner by overexpression of HA-TAZ in IMCD cells expressing Flag-PKD1. IMCD cells were cotransfected with the indicated amounts of HA-TAZ plasmids and Flag-PKD1. (C and D) Luciferase assays for β-catenin activity. IMCD cells expressing the indicated vectors were transfected with pTOP Flash and Renilla luciferase vectors. (E) Inhibition of the reduction of active MEDICAL SCIENCES β-catenin protein levels. IMCD cells expressing siRNA against AXIN1 or control transfected with the indicated plasmids. (F and G) Luciferase assays for β-catenin activity in the cells transfected or transduced with the indicated siRNAs, plasmids, shTAZ, or expression plasmids. (H) qRT-PCR analysis of β-catenin target genes AXIN1, c-MYC, and CCND2. Cells were transduced with the indicated siRNAs or shTAZ retroviral vectors.

with Myc-AXIN1 in the si-control cells, whereas a lesser copre- Conversely, AXIN1 immunoprecipitates in Pkd1 knockout mouse cipitation of β-catenin and AXIN1 was observed in PKD1- kidney revealed a significant interaction with TAZ compared to depleted cells (Fig. 6B). Moreover, Myc-AXIN1 slightly copre- that of the wild type kidney. Interestingly, the interaction between cipitated TAZ in si-control cells, but strongly interacted with β-catenin and AXIN1 was decreased to a lesser extent in Pkd1 B TAZ in the absence of PKD1 (Fig. 6 ). We also tested the ef- mutant mouse kidney (Fig. 6G). Taken altogether, deficiency of β fects of excess PKD1 on the association of AXIN1 with -catenin PKD1 leads to a strong interaction of TAZ with AXIN1, and or TAZ. We observed that AXIN1 coimmunoprecipitated well β-catenin is thereby released from the destruction complex and is with TAZ or β-catenin in the control cells but interacted weakly transcriptionally activated. with TAZ and had stronger interaction with β-catenin (Fig. 6C). These results indicated that PKD1 depletion renders TAZ prone Colocalization of TAZ and AXIN1 in Pkd1-Silenced Cells. Polycystin-1 to interaction with AXIN1, resulting in a weak interaction of and YAP/TAZ have been proposed to be mechanosensing pro- β-catenin with AXIN1. On the contrary, excess PKD1 did not allow TAZ to interact with AXIN1, consequently enhancing the teins. A recent study suggested that overexpression of PKD1 interaction with β-catenin. To further assess whether abundant decreased nuclear localization of YAP (19). We hypothesized TAZ levels affected the interaction of AXIN1 with β-catenin, we that TAZ localization is regulated by the PKD1 aspect of their carried out IP with HA-TAZ–expressing cells. Immunoprecipi- role in mechanosensing. At 50% cell density, TAZ is mostly tation of endogenous AXIN1 revealed β-catenin, whereas forced localized in the , with some in the nucleus (Fig. 7A). expression of HA-TAZ induced a weak interaction of AXIN1 Meanwhile, PKD1-silenced cells displayed an increase of nuclear with β-catenin compared to the control (Fig. 6D). β-Catenin TAZ (Fig. 7A). It suggested that the presence of PKD1 retained released from the destruction complex localizes to the nucleus TAZ in the cytoplasm. to activate the target genes. We therefore determined whether Previously, we found that the interaction of TAZ with AXIN1 β-catenin unbound from AXIN1 was present in the nucleus of is increased in the absence of PKD1. We examined whether TAZ – HA-TAZ expressing cells. Cells used for the experiments pre- is colocalized with AXIN1 in the absence of PKD1. Ectopically D β sented in Fig. 6 were used for nuclear fractionation. -Catenin expressed HA-TAZ was detected in the cytoplasm of control – was observed in the nuclear fraction of HA-TAZ expressing cells, whereas PKD1-silenced cells showed an increased HA- cells. Notably, HA-TAZ was also present in the same fraction TAZ nuclear translocation, implying PKD1-dependent cyto- (Fig. 6E). To determine whether TAZ interacts with intact plasmic localization of TAZ (Fig. 7B). PKD1 knockdown in- AXIN1 in Pkd1-null mouse kidney, we prepared tissue lysates f/f f/f from Pkd1 or Pkd1 ;HoxB7cre mice kidney. TAZ or AXIN1 creased the interaction of TAZ with AXIN1. We investigated was immunoprecipitated and then blotted for AXIN1, PKD1, and whether HA-TAZ expression colocalized with Myc-AXIN1 in active β-catenin (Fig. 6 F and G). Consistent with previous results, PKD1-silenced cells. PKD1 knockdown led the colocalization of a weak interaction of TAZ with AXIN1 was shown in wild type HA-TAZ with Myc-AXIN1 in the cytoplasm (Fig. 7B), suggest- f/f mouse kidney, whereas Pkd1 ;HoxB7cre mouse kidney revealed ing that PKD1 depletion induced colocalization of HA-TAZ increased interaction between AXIN1 and TAZ (Fig. 6F). with Myc-AXIN1.

Lee et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 29007 Downloaded by guest on September 24, 2021 Fig. 6. A strong interaction of AXIN1 with TAZ in the absence of PKD1 causes the activation of β-catenin. (A) Interaction of HA-TAZ with AXIN1 in the absence of PKD1. HA-TAZ was cotransfected with si-control or si-PKD1 into inner medullary collecting duct (IMCD) cells, and immunoprecipitation was performed with an anti-HA antibody, followed by immunoblotting with the indicated antibodies. (B) A strong interaction of MYC-AXIN1 with HA-TAZ in PKD1-silenced cells. The experiments were performed as described for A. MYC-AXIN1 was cotransfected with the indicated siRNAs. (C) A weak interaction of MYC-AXIN1 with HA-TAZ upon overexpression of PKD1. Flag-PKD1 or control vectors were transfected, and endogenous AXIN1 was immunoprecipitated with an anti-AXIN1 antibody. (D and E) A weak interaction of AXIN1 with β-catenin by forced expression of HA-TAZ. Half of HA-TAZ–transfected cells were used for immunoprecipitation as described in C, and the remaining cells were used for nuclear fractionation (E). All immunoprecipitations with cell lysates were performed in at least two independent experiments. (F and G) A strong interaction of AXIN1 with TAZ in Pkd1-null kidney. Lysates prepared from Pkd1f/f mouse kidney or Pkd1f/f;HoxB7 cre kidney were subjected to immunoprecipitation using TAZ- or AXIN1-specific antibody and IgG as control.

c-MYC Transcription Is Increased by TAZ and β-Catenin in reduced in PKD1/TAZ-codepleted cells and in TAZ-depleted PKD1-Depleted Cells. We found that silencing of PKD1 in cells cells (Fig. 8B). Consistent with these results, c-MYC levels led to the transcriptional activation of β-catenin and that ex- were significantly higher in PKD1-silenced cells but were re- pression of c-MYC was increased in the kidney of Pkd1-null duced in the codepleted cells or in TAZ-depleted cells (Fig. 8C). mice. Furthermore, c-MYC is a known target gene of β-catenin These results suggest that TAZ is responsible for the expression or YAP/TAZ and is implicated in the pathogenesis of cystic of c-MYC in PKD1-depleted cells. We further tested whether kidney (9, 11). Thus, we proposed that the deletion of PKD1 increased β-catenin activity in PKD1-silenced cells contributed to contributed to the induction of c-MYC expression during cys- the expression of the c-MYC gene. As expected, increased togenesis. We first investigated whether β-catenin or TAZ was c-MYC expression under PKD1-deficient condition was reduced localized to the nucleus to regulate the transcription of the by the knockdown of β-catenin (Fig. 8D). Moreover, the effect of c-MYC gene. We performed cellular fractionation of PKD1- β-catenin knockdown on c-MYC expression was less than that of depleted cells (Fig. 8A). As expected, the amount of active TAZ depletion, indicating that the expression of c-MYC mRNA β-catenin protein was high in the nuclear fraction of PKD1- remained regulated by TAZ. depleted cells (Fig. 8A). Interestingly, TAZ was partially pre- Overall, based on our results, it can be suggested that the sent in the nuclear fraction of PKD1-depleted cells, and TAZ depletion of TAZ in PKD1-silenced cells reduces the expression phosphorylated at serine 89 was consistently decreased slightly in of c-MYC mRNA via TAZ-mediated regulation of β-catenin or the cytosolic fraction of these cells (Fig. 8A). Our findings indi- TAZ (Fig. 9). cate that PKD1 depletion induces the translocation of active β-catenin or TAZ into the nucleus, suggesting that this may Discussion participate in an increase in the expression of c-MYC. To test We have demonstrated a role for TAZ in the regulation of this hypothesis, we performed qRT-PCR to determine the ex- β-catenin in the pathogenesis of ADPKD. Highly accumulated pression of c-MYC mRNA in PKD1- or TAZ-depleted cells or in TAZ was observed in cyst-lining epithelium of the collecting cells codepleted for PKD1 and TAZ. The silencing of PKD1 duct-targeted Pkd1 knockout mice kidneys with high expression increased the expression of c-MYC mRNA, which was markedly of c-MYC and active β-catenin. The deficiency of TAZ rescued

29008 | www.pnas.org/cgi/doi/10.1073/pnas.2009334117 Lee et al. Downloaded by guest on September 24, 2021 Fig. 7. Colocalization of AXIN1 and TAZ in the ab- sence of PKD1. Immunofluorescence staining of MYC-AXIN1 (red) and HA-TAZ (green) in PKD1 knockdown IMCD cells. (A) siRNAs against control or PKD1 were delivered into cells, and then HA-TAZ was transfected and immunostained for HA-TAZ and nuclei counterstained with DAPI. Knockdown of PKD1 with siPKD1 increased nuclear localization of HA-TAZ. The distribution of HA-TAZ was scored as cytoplasmic (“C”) and cytoplasmic and nuclear (CN) and graphed. The results are the average of three independent experiments; at least 100 cells were scored for each sample in every experiment. (B) Pkd1-depleted cells were transfected with MYC- AXIN1 and HA-TAZ. PKD1 knockdown increased colocalization of HA-TAZ and MYC-AXIN1 in the cy- toplasm. (Scale bar, 20 μm.) MEDICAL SCIENCES

renal cyst formation with reduced active β-catenin and c-MYC in increased in Pkd1-knockout kidneys; 2) Pkd1 knockout mice Pkd1-null mice. Likewise, the formation of in vitro cysts stimu- developed polycystic kidneys, but this was rescued by the dele- lated upon forskolin treatment was reduced by Taz depletion in tion of Taz; and 3) increased cystic growth in Pkd1-silenced cells Pkd1-silenced cells. Our results prove that TAZ participates in under forskolin treatment was restored to the basal levels by the activation of β-catenin, as PKD1 deficiency renders TAZ TAZ knockdown. prone to interact with AXIN1, resulting in a weak interaction of YAP and TAZ are believed to play distinct roles in regulating β-catenin with AXIN1. β-Catenin and TAZ were detected in the the expression of target genes despite their functional redun- nucleus, where they regulated the c-MYC expression. Conse- dancy and similarities in their protein sequence. For example, quently, we suggest two independent roles of TAZ in the regu- loss of TAZ leads to cystic proximal tubule in a normal-sized lation of c-MYC mRNA implicated in cystogenesis in the kidney kidney whereas the YAP mutant shows reduced nephrogenesis of Pkd1-depleted mice: 1) β-catenin activation via the TAZ– (27). RNA-seq analyses revealed that many of the genes that are AXIN1 axis or 2) transcriptional activation of nuclear TAZ itself. differentially expressed either by YAP or TAZ are key tran- YAP and TAZ, the paralogous effectors of the Hippo path- scription factors or growth factors (26). It is therefore believed way, are known to be involved in PKD; however, their roles are that context-dependent binding to DNA-binding partners, such complicated. Both have been reported to be commonly activated as Pax3, TBX-5, TTF1, and PPAR gamma for TAZ (28–31) and around the cystic epithelium and are involved in regulating cyst ErbB4 and for YAP (32, 33), endows YAP and TAZ with growth (8, 20, 21). A recent study showed that loss of YAP in their respective regulatory traits. It has been reported that YAP Pkd1-deleted mice alleviated the PKD phenotypes, and negatively regulates TAZ at the protein level, indicating that LARG-RhoA-ROCK signaling module mediated the increase in TAZ might influence other downstream signaling pathways in YAP/TAZ, followed by c-MYC activation in the Pkd1-deleted the cytoplasm irrespective of YAP (24, 34, 35). In our study, the model. These authors also observed that transgenic expression of levels of TAZ proteins were more faithfully increased than YAP YAP enhanced the cell proliferation and dilation of tubules in in the kidneys of Pkd1-null mice. This evidence provides a strong the kidneys (9). This is in contrast to the observation that renal clue indicating a potential link between TAZ and PKD1 in renal cysts appeared in Taz-deficient mice with tubule dilation. Inac- cystogenesis in comparison with the link between YAP1 and tivation of TAZ results in the development of renal cysts pre- PKD1. As expected, TAZ depletion alleviated the Pkd1 mutant dominantly originating from the glomerulus cells, finally leading phenotype and inhibited the increase in c-MYC expression in the to end-stage renal disease (ESRD) (22). In another study on Taz- kidneys of Pkd1-deleted mice or Pkd1-silenced cells. We con- deficient mice, multicystic kidneys with other pathologic phe- cluded that TAZ functions as a downstream signal of PKD1, notypes, including urinary concentration defects, were observed although the role of YAP remains to be determined. (23). In addition to the conflicting effects of the lack of either Previous studies have suggested the possibility that the up- YAP or TAZ on the formation of renal cysts in PKD rodent stream regulator of YAP/TAZ could affect the activation of models, increasing evidence has highlighted the independent Wnt/β-catenin signaling. Some studies have reported that the roles of each protein (24–26). Our observations support the Hippo pathway YAP/TAZ antagonizes the Wnt/β-catenin sig- notion that TAZ contributes to the disease progression in Pkd1- naling via sequestering the β-catenin in the destruction complex depleted kidneys based on the following evidence: 1) TAZ was (17, 36, 37). In colorectal , cytosolic YAP/TAZ has been

Lee et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 29009 Downloaded by guest on September 24, 2021 Fig. 8. Increase in the expression of c-MYC in Pkd1-deficient cells. (A) Localization of active β-catenin and TAZ in the nucleus by PKD1 knockdown. Cells were transfected with si-control or si-PKD1 RNAs and subsequently lysed with a hypotonic buffer to prepare the cytoplasmic fraction and nuclear pellets, which were lysed with RIPA buffer. (B and C) Inhibition of the increase in c-MYC mRNA and protein levels in codepleted cells transfected with siPKD1 RNAs or shTAZ retroviral vectors. Following transduction of shTAZ retroviral vectors or siPKD1 RNA transfection, qRT-PCR and Western blot analyses were performed. (D)The regulation of c-MYC protein levels by TAZ and β-catenin. Western blot analysis was performed for the indicated proteins.

reported to inhibit the nuclear activity of β-catenin either via suggest a potential therapeutic strategy that combines Wnt- direct binding or by association with the destruction complex targeting drugs with mTOR inhibition to delay disease progres- comprising AXIN1 (17, 38). However, our experiments with sion in ADPKD. Pkd1/Taz-double-knockout mice suggested that TAZ triggered In conclusion, our data show that the PKD1–TAZ–Wnt–β- the β-catenin activation via interaction with AXIN1, thereby catenin–c-MYC signaling axis regulates renal cystogenesis, which leading to an increase in the expression of c-MYC. In line with might be a potential therapeutic target against ADPKD. our findings, evidence exists to support that YAP/TAZ activates the Wnt/β-catenin signaling. Sav and Mst1/2 depletion in neo- Materials and Methods natal heart enhanced the expression of target genes controlled by . Pkd1-floxed mice containing loxP sites flanking the exons 2 to 5 of the Wnt/β-catenin signaling (36). Also, we previously reported that Pkd1 gene were obtained from Stefam Somlo, Yale University, New Haven, the activation of TAZ in Sav1-knockout mice was accompanied CT (42). Taz-floxed mice, containing Taz mutant allele in which exon 3 is β replaced by one loxP site, established at Korea Advanced Institute of Science by elevated -catenin expression (12). Recently, inflammation- and Technology, were provided by Dae-Sik Lim, Korea Advanced Institute of mediated regeneration of intestinal epithelial cells increased the Science and Technology, Daejeon, Korea, and transferred and bred in spe- nuclear colocalization of YAP and β-catenin (38). Therefore, a cific pathogen-free facilities at Sookmyung Women’s University (10, tissue-specific or context-dependent relationship between β-catenin 43). HoxB7Cre transgenic mice, which drive cre-recombinase activity specif- and YAP/or TAZ is anticipated. ically in the renal collecting duct, were used. All mice were maintained Wnt/β-catenin signaling has been reported to be involved in in C57BL6 genetic background and bred in an animal facility under opti- cystogenesis in renal cystic disease. However, the role of Wnt/ mal conditions (12-h light/dark at 20 °C) following the approval of ’ β-catenin signaling in renal cystogenesis remains unclear. High the institutional animal care and use committee at Sookmyung Women s University. levels of β-catenin were reported to induce cyst formation in the kidney (13, 39). Our findings also indicated that the activation of β Human Kidney Specimens. As a control group, normal regions of human -catenin by an interaction of TAZ and AXIN1 was responsible kidneys, confirmed by histologic examination, were acquired from renal cell for renal cystogenesis in Pkd1 knockout mice. Therefore, we carcinoma patients undergoing surgical treatment. All biological samples and postulated that the TAZ/AXIN1/β-catenin axis could be a ther- data were deidentified prior to use in this study. Informed consent was apeutic target against ADPKD. Similar to our suggestions, a waived because of the retrospective nature of the study and because the study reported that inhibition of Wnt signaling by Wnt biogenesis analysis used anonymous clinical data. The study was approved by the medical inhibitor LGK974 delays the progression of renal cystogenesis ethics committee of St. Vincent’s Hospital (VC20SISI0136). (40). Our data also revealed that Endo IWR1, which inhibited β-catenin activity via AXIN stabilization, reduced in vitro cyst Histology and Immunofluorescence Analyses. Paraffin-embedded mice kidneys were cut into 6-μm sections and stained with hematoxylin and eosin (H&E) for growth in Pkd1-silenced cells. It also showed a significant de- histological analysis. Tissue slides were also probed with antibodies against crease in in vitro cysts when Endo IWR1 was coadministered TAZ (Abcam), c-MYC (Santa Cruz, sc-764), β-catenin (Millipore, 05–665), DBA with rapamycin, which previously had been reported to inhibit (Vector, FL-1031), Ki-67 (Abcam, ab16667), and collagen IV (Southern Biotech, renal cysts in preclinical models of ADPKD (41). These data 1340–01) to monitor the expression of the respective target proteins.

29010 | www.pnas.org/cgi/doi/10.1073/pnas.2009334117 Lee et al. Downloaded by guest on September 24, 2021 Fig. 9. Schematic representation of the regulation of TAZ/Wnt-β-catenin/c-MYC axis by PKD1 in renal cystogenesis. TAZ interacts with PKD1. Cytosolic β-catenin is located in the destruction complex composed of AXIN1, APC, and β-TrCP. In PKD1-deleted cells, TAZ is triggered to interact with AXIN1 and β-catenin is released from the destruction complex to localize in the nucleus. A part of TAZ is also present in the nucleus, where it induces the expression of c-MYC mRNA. MEDICAL SCIENCES

Immunofluorescence of IMCD Cells. Cells were fixed for 20 min, permeabilized Antibodies. The sources and catalog numbers of the antibodies used were as with 0.4% Triton X-100/PBS for 20 min, and blocked with 5% BSA in PBST follows: PC1 (sc-130554), c-MYC (sc-40), HA (sc-7392), AXIN1 (sc-293190), (PBS plus 0.05% Tween-20) for 30 min at room temperature. Primary anti- β-catenin (sc-7963), p-TAZ (Ser89; sc-17610), and alpha tubulin (sc-5286; all bodies (anti-HA mouse mono antibody and anti-Myc rabbit poly antibody) from Santa Cruz Biotechnology); HA (ab969110), Myc (ab9106), and Flag were applied for 1 h at room temperature, washed with PBST at least three (ab1257; all from Abcam); YAP/TAZ (no. 8418), α-tubulin (no. 2144), AXIN1 times, and visualized using Alexa Fluor 488 donkey anti-mouse or anti-rabbit (no. 2087), c-MYC (no. 9402), phospho-CREB (no. 9198), Histone H3 (no. 568-conjugated secondary antibodies (Invitrogen) used at 1:200 for 1 h at 60538), and LSD1 (Lysine-specific demethylase 1; no. 2139; all from Cell room temperature. Preparations were mounted in Dako mounting medium Signaling); β-actin (A2228; Sigma); β-catenin (no. 610153) and TAZ (no. containing DAPI (Vector Laboratories). 560235; both from BD Biosciences); and anti–active-β-catenin (ABC 05–665; Millipore). For immunofluorescence, anti-HA tag antibody (ab18181; Abcam) Cell Culture and Reagents. Mouse inner medullary collecting duct (IMCD) cells and anti-Myc tag rabbit (sc-2278; ) were used. were cultured in Dulbecco’s modified Eagle’s medium F/12 (DMEM F/12; Welgene) supplemented with 10% fetal bovine serum (FBS). For in vitro qRT-PCR. RNA was extracted from cell lysates and mouse kidney samples using × 5 analysis of cysts, 2.0 to 3.0 10 cells/mL of DMEM F/12 were plated on NucleoSpin RNA/Protein column (Macherey-Nagel) and reverse-transcribed Matrigel (Corning, no. 354230) at a 1:1 ratio and cultured by addition of with M-MLV reverse transcriptase (Promega) according to the manufac- DMEM F/12 without FBS for 5 to 6 d. The drugs, including forskolin turers’ protocols. The cDNA was mixed with SYBR Green and primers for the (Sigma-Aldrich, no. F3917), TAZ modulator (Merck, no. 530959), Exo IWR1 target genes. The following primers were used: Pkd1 (F: 5′-GGGAGCCCTGCC- (Tocris, no. 3947), and Endo IWR1 (Tocris, no. 3532), were used at optimal ACCTACCTA-3′,R:5′-CCTCACTACGGCTCACCTCATTCC-3′), TAZ (F: 5′-AGTTCC- concentrations. Exo- and Endo-IWR1 were provided by E.J. For 3D culture, TGCGCTTCAAATGGG-3′,R:5′-GTAGGGTGGGCTGTTAGGGAG-3′), AXIN2 (F: drugs were added to the medium at least 2 d after seeding, and the treat- 5′-TACACTCCTTATTGGGCGATCA-3′,R:5′-TTGGCTACTCGTAAAGTTTTGGT- ment was continued until the in vitro cysts had grown sufficiently. 3′), c-MYC (F: 5′-GGCTCCTGGCAAAAGGTCA-3′,R:5′-CTGCGTAGTTGTGCT- GATGT-3′), CCND2 (F: 5′-ACCTTCCGCAGTGCTCCTA-3′,R:5′-CCCAGCCAAGAA- Gene Knockdown, Expression Constructs, and Transfection. The cells were ACGGTCC-3′), Ankrd1 (F: 5′-GCTGCGCTGGAGAACAAACTG-3′,R:5′-AGCCTCCAT- transfected either with a scrambled siRNA (Santa Cruz) or with an siRNA targeting TAACTTCTCCACGAT-3′), Fgf5 (F: 5′-TTTTCTTCGTCTTCTGCCTCCTCA-3′,R: Pkd1 (Santa Cruz, sc-40862), TAZ (Santa Cruz, sc-38569), or AXIN1 (Santa Cruz, sc- 5′-GAAACCGATGCCCACTCTGC), Foxf2 (F: 5′-CGGCGCCTCTGGGTTGC-3′,R: 41449) using Lipofectamine RNAiMAX (Invitrogen) for at least 24 h. Flag-PKD1 5′-GGTGGTGGTGGAGGTGGTGTG-3′), Gadd45d (F: 5′-GCACTGCCTCCTGGT- was purchased from Addgene (cat. no. 31793). HA-TAZ plasmid was a gift from CACGAA-3′,R:5′-CCCATTGGTTATTGCCTCTGCTCT-3′), and Serpine1 (F: 5′- Dae-Sik Lim. HA–β-catenin and Myc-AXIN1 were provided by E.J. The cells were ′ ′ ′ transfected with these expression plasmids using Lipofectamine 2000 for 24 h. GCAACCCTGGCCGACTTCA-3 ,R:5-ACGCCACTGTGCCGCTCTC-3 ).

Western Blot Analysis and Immunoprecipitation. Protein lysates from mouse RNA-Seq Data Analysis. For identification of transcriptome levels in Pkd1- tissues were purified either by using NucleoSpin RNA/Protein column deleted kidneys compared to controls, RNA sequencing data from GSE86509 (Macherey-Nagel) or nucleus/cytosol fractionation (Thermo). Subcellular was reanalyzed, which had been done in our previous study and published on fractionation was obtained using a homogenizer as previously described (44). the GEO public database (https://www.ncbi.nlm.nih.gov/geo/) (15). GSEA (gene Western blot and immunoprecipitation (IP) with cell lysates were performed in set enrichment analysis) was conducted to assure differentially expressed gene at least two independent experiments as previously described (44). Pkd1f/f and levels between Pkd1-deleted kidney tissues and controls (45). Pkd1f/f;HoxB7cre mouse kidney were used for Axin or Taz immunoprecipitates. Briefly, mouse kidney tissue fragments were homogenized using a pestle in Luciferase Assay. pTOP reporter constructs and pTk-Renilla (transfection 200 μL of cold lysis buffer containing 1% Triton X-100. Homogenates were control; ratio of DNA amounts was 1:10) were cotransfected into IMCD cells centrifuged at 12,000 rpm (20 min, 4 °C), and the supernatant was used for expressing the indicated plasmid or siRNA oligonucleotides. Luciferase ac- preclearing in IP buffer containing normal rabbit IgG and A/G agarose beads. tivity was measured using the Promega luciferase assay reagents as de- Precleared supernatant was used for IP. scribed by the manufacturer.

Lee et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 29011 Downloaded by guest on September 24, 2021 Viral Vectors. For knockdown of human TAZ or human β-catenin, shRNA Data Availability. All study data are included in the article and supporting vectors against TAZ or β-catenin were delivered using a retroviral vector information. containing a puromycin resistance cassette. The viral particles were pro- duced as previously described (17). shRNA against β-catenin and TAZ retro- ACKNOWLEDGMENTS. This work was supported by grants from the Bio & Medical Technology Development Program (2015M3A9B6027555) and the viral vectors were provided by E.J. and Dae-sik Lim. Basic Science Program (2016R1A5A1011974) and was also supported by Basic Science Research Program through the National Research Foundation of Statistical Analysis. All of the data were obtained from a minimum of three Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2018R1A2B6003440 and NRF2016R1D1A1B01012138). This research independent experiments and were statistically analyzed as means ± SD. Un- was also supported by the Bio & Medical Technology Development Program paired t test was performed using GraphPad Prism 5.0 (GraphPad software). of the National Research Foundation (NRF) funded by the Korean govern- Results with a P value less than 0.05 were considered statistically significant. ment (MSIT; no. 2019M3A9H110376911).

1. A. C. Ong, P. C. Harris, Molecular pathogenesis of ADPKD: The polycystin complex gets 26. S. W. Plouffe et al., The Hippo pathway effector proteins YAP and TAZ have both complex. Kidney Int. 67, 1234–1247 (2005). distinct and overlapping functions in the cell. J. Biol. Chem. 293, 11230–11240 (2018). 2. P. C. Harris, V. E. Torres, Polycystic kidney disease. Annu. Rev. Med. 60, 321–337 (2009). 27. A. Reginensi et al., Yap- and Cdc42-dependent nephrogenesis and morphogenesis 3. C. G. Hansen, T. Moroishi, K.-L. Guan, YAP and TAZ: A nexus for Hippo signaling and during mouse kidney development. PLoS Genet. 9, e1003380 (2013). – beyond. Trends Cell Biol. 25, 499 513 (2015). 28. M. Murakami, M. Nakagawa, E. N. Olson, O. Nakagawa, A WW domain protein TAZ is 4. W. Hong, K.-L. Guan, The YAP and TAZ transcription co-activators: key downstream a critical coactivator for TBX5, a implicated in Holt-Oram syn- effectors of the mammalian Hippo pathway. Semin. Cell Dev. Biol. 23, 785–793 (2012). drome. Proc. Natl. Acad. Sci. U.S.A. 102, 18034–18039 (2005). 5. X. Varelas, The Hippo pathway effectors TAZ and YAP in development, homeostasis 29. M. Murakami et al., Transcriptional activity of Pax3 is co-activated by TAZ. Biochem. and disease. Development 141, 1614–1626 (2014). Biophys. Res. Commun. 339, 533–539 (2006). 6. W. Choi et al., YAP/TAZ initiates gastric tumorigenesis via upregulation of MYC. 30. K.-S. Park et al., TAZ interacts with TTF-1 and regulates expression of surfactant Cancer Res. 78, 3306–3320 (2018). – 7. D. Xu et al., Scribble influences cyst formation in autosomal-dominant polycystic protein-C. J. Biol. Chem. 279, 17384 17390 (2004). kidney disease by regulating Hippo signaling pathway. FASEB J. 32, 4394–4407 (2018). 31. J.-H. Hong et al., TAZ, a transcriptional modulator of mesenchymal stem cell differ- – 8. H. Happé et al., Altered Hippo signalling in polycystic kidney disease. J. Pathol. 224, entiation. Science 309, 1074 1078 (2005). 133–142 (2011). 32. J. W. Haskins, D. X. Nguyen, D. F. Stern, Neuregulin 1-activated ERBB4 interacts with 9. J. Cai et al., A RhoA-YAP-c-Myc signaling axis promotes the development of polycystic YAP to induce Hippo pathway target genes and promote cell migration. Sci. Signal. 7, kidney disease. Genes Dev. 32, 781–793 (2018). ra116 (2014). 10. H. Cho et al., YAP and TAZ negatively regulate Prox1 during developmental and 33. D. Levy, Y. Adamovich, N. Reuven, Y. Shaul, The Yes-associated protein 1 stabilizes pathologic lymphangiogenesis. Circ. Res. 124, 225–242 (2019). p73 by preventing Itch-mediated ubiquitination of p73. Cell Death Differ. 14, 743–751 11. M. Trudel, V. D’Agati, F. Costantini, C-myc as an inducer of polycystic kidney disease in (2007). transgenic mice. Kidney Int. 39, 665–671 (1991). 34. M. L. Finch-Edmondson et al., TAZ protein accumulation is negatively regulated by 12. E. Seo et al., The Hippo-Salvador signaling pathway regulates renal tubulointerstitial YAP abundance in mammalian cells. J. Biol. Chem. 290, 27928–27938 (2015). fibrosis. Sci. Rep. 6, 31931 (2016). 35. S. Muppala, V. K. Raghunathan, I. Jalilian, S. Thomasy, C. J. Murphy, YAP and TAZ are 13. V. Patel, Balancing the Wnts in polycystic kidney disease. Am. Soc. Nephrol. 21, distinct effectors of corneal myofibroblast transformation. Exp. Eye Res. 180, 102–109 1412–1414 (2010). (2019). 14. M. A. Lancaster et al., Impaired Wnt-β-catenin signaling disrupts adult renal ho- 36. T. Heallen et al., Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte meostasis and leads to cystic kidney ciliopathy. Nat. Med. 15, 1046–1054 (2009). proliferation and heart size. Science 332, 458–461 (2011). 15. Y. M. Woo et al., Profiling of miRNAs and target genes related to cystogenesis in 37. X. Varelas et al., The Hippo pathway regulates Wnt/beta-catenin signaling. Dev. Cell ADPKD mouse models. Sci. Rep. 7, 14151 (2017). – 16. B. Chen et al., Small molecule-mediated disruption of Wnt-dependent signaling in 18, 579 591 (2010). β tissue regeneration and cancer. Nat. Chem. Biol. 5, 100–107 (2009). 38. F. Deng et al., YAP triggers the Wnt/ -catenin signalling pathway and promotes en- 17. L. Azzolin et al., YAP/TAZ incorporation in the β-catenin destruction complex or- terocyte self-renewal, regeneration and tumorigenesis after DSS-induced injury. Cell chestrates the Wnt response. Cell 158, 157–170 (2014). Death Dis. 9, 153 (2018). 18. Z. Xiao et al., Polycystin-1 interacts with TAZ to stimulate osteoblastogenesis and 39. S. Saadi-Kheddouci et al., Early development of polycystic kidney disease in transgenic inhibit adipogenesis. J. Clin. Invest. 128, 157–174 (2018). mice expressing an activated mutant of the β-catenin gene. 20, 5972–5981 19. E. A. Nigro et al., Polycystin-1 regulates actomyosin contraction and the cellular re- (2001). sponse to extracellular stiffness. Sci. Rep. 9, 16640 (2019). 40. A. Li et al., Canonical Wnt inhibitors ameliorate cystogenesis in a mouse ortholog of 20. V. Grampa et al., Novel NEK8 mutations cause severe syndromic renal cystic dysplasia human ADPKD. JCI Insight 3, e95874 (2018). through YAP dysregulation. PLoS Genet. 12, e1005894 (2016). 41. H. J. Kim, C. L. Edelstein, Mammalian target of rapamycin inhibition in polycystic 21. L. Jiang et al., Increased YAP activation is associated with hepatic cyst epithelial cell kidney disease: From bench to bedside. Kidney Res. Clin. Pract. 31, 132–138 (2012). – proliferation in ARPKD/CHF. Gene Expr. 17, 313 326 (2017). 42. S. Shibazaki et al., Cyst formation and activation of the extracellular regulated kinase 22. Z. Hossain et al., Glomerulocystic kidney disease in mice with a targeted inactivation pathway after kidney specific inactivation of Pkd1. Hum. Mol. Genet. 17, 1505–1516 of Wwtr1. Proc. Natl. Acad. Sci. U.S.A. 104, 1631–1636 (2007). (2008). 23. R. Makita et al., Multiple renal cysts, urinary concentration defects, and pulmonary 43. M. Xin et al., Hippo pathway effector Yap promotes cardiac regeneration. Proc. Natl. emphysematous changes in mice lacking TAZ. Am. J. Physiol. Renal Physiol. 294, – F542–F553 (2008). Acad. Sci. U.S.A. 110, 13839 13844 (2013). 24. C. Sun et al., Common and distinctive functions of the Hippo effectors Taz and Yap in 44. E. Seo et al., regulates YAP1 to maintain stemness and determine cell fate in the – skeletal muscle stem cell function. Stem Cells 35, 1958–1972 (2017). osteo-adipo lineage. Cell Rep. 3, 2075 2087 (2013). 25. J. Xiong, M. Almeida, C. A. O’Brien, The YAP/TAZ transcriptional co-activators have 45. A. Subramanian et al., Gene set enrichment analysis: A knowledge-based approach opposing effects at different stages of osteoblast differentiation. Bone 112,1–9 for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A. 102, (2018). 15545–15550 (2005).

29012 | www.pnas.org/cgi/doi/10.1073/pnas.2009334117 Lee et al. Downloaded by guest on September 24, 2021