Page 49 - ebook

Deficiency of TAZ reduced ciliogenesis in renal glomeruli

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Jae Hee Jun , Minah Park , Gyuyeong Rha, Yejin Ahn, Jin-ui Min, Jong Hoon Park 1,*
1 Department of Biological Science, Sookmyung Women’s University, Seoul, 04310; Republic of Korea Cell Biology (B-24)
Abstract
The primary cilia are antenna-like organelles and transmit the extracellular signals to cell that maintain cellular homeostasis with the Shh, Wnt, Notch,
and mTOR signaling pathways. In the case of ciliary defect, there are various diseases polydactyly, sensorineural deafness, renal fibrosis and
inflammation, cystic renal disease, and retinal degeneration without cilia in sensory organs such as kidney, brain, and pancreas, etc. Thus, research for
following to regulate primary cilia is important. The most representative ciliary defect case in kidney is polycystic kidney disease. This disease forms cysts
full of fluid and loses renal function along with various complications, which requires continuous dialysis or kidney transplantation. Recent studies have
revealed that the hippo signaling is associated with primary cilia and cystic diseases, and YAP, hippo signaling key regulator, involved in ciliogenesis by
modulating actin remodeling factors.
In this study, we investigated the mechanism by TAZ deficiency which is orthologous YAP, affects primary cilia formation in each renal tubule type. Little
is known about the function of TAZ regulating ciliogenesis and independent from YAP. We discovered TAZ defect reduced primary cilia only in glomerular
cells not renal collecting duct cells or proximal tubule cells. Although our study focused on TAZ, ciliogenesis, and other ciliary genes, the results suggest
the distinct roles of YAP and TAZ, specifically in terms of ciliary regulation in kidneys tubules.
Scheme Fig. 3. Different roles of YAP and TAZ in cilia formation in multiple types
of renal tubular cells
Cyst and Cilia phenotypes in TAZ defect mouse model (in vivo) (a-l) Observations of
• Screening of cyst formation and primary cilia phenotypes in Taz- fluorescently labeled primary
floxed:KSP-cre and Taz-floxed:HoxB7-cre mice. cilia after silencing of either YAP
• Differential TAZ expression in specific renal tubules in WT
or TAZ in three different types of
cells—mIMCD, collecting duct;
Relationship between TAZ and cilia SV40MES13, glomeruli; and
TAZ expression changes during ciliogenesis with NIH/3T3 TKPTS, proximal tubule. Cells
were then serum starved for at
least 24 h. (b, f, j) The graphs
Opposite roles of YAP and TAZ in ciliogenesis depending on the renal show the ratio of ciliated cells
tubular cell type and (c, g, k) cilia lengths were
• Positive regulation of TAZ with ciliogenesis in SV40MES13 cell. measured from the basal body
• No significant regulation of TAZ with ciliogenesis in IMCD cell. to the ciliary tip and (d, h, l) the
• Negative regulation of TAZ with ciliogenesis in TKPTS cell. proportions of cilia lengths in
three ranges: <2.5 µm, 2.5 - 5
μm, and >5 μm.
Validation of ciliary genes that change with decreased TAZ expression Fig. 4. IFT140, NPHP6, NPHP9 changed with decreased TAZ expression
•IFT140 decrease and abnormal localization with cilia under TAZ K/D
•Primary cilia recovery by reducing NPHP 6 & 9 increased by TAZ K/D (a) IFT140 protein expression
with YAP or TAZ silencing and
Result subjected to 24 hours of serum
starvation. (b, C) ICC of IFT140 (red)
Fig. 1. Cyst formation and cilia reduction in Taz-floxed:KSP-cre and and basal body (green) and the
different localization of TAZ in specific renal tubules graph shows localization of IFT140
in primary cilia was classified into
(a) Renal tissue sections 4 categories. (d) NPHP6 & 9 mRNA
from WT, TAZ cKO and TAZ- expression changed with identical
CD cKO) mice at 36 weeks (b) conditions. (e, f, g, h)
Fluorescence staining of Accumulation of NPHP6 & 9 (red)
tubule markers. LTL, in primary cilia (green) under
proximal tubule; Calbindin, identical conditions and the
distal convoluted tubule; graphs show NPHP 6 & 9
THP, ascending loop of fluorescence intensities localized
Henle; DBA, collecting duct. around primary cilia. (i-l) Primary
(c) The ratio of 2KW/TBW in cilia recovery in cells with TAZ
WT, TAZ cKO, and TAZ CD- silencing and knockdown of either
cKO mice at 36 weeks. NPHP 6 or 9 after 24 h of serum
(d) Graph quantifying cilia-positive DBA labeling in WT, TAZ CD-cKO and starvation. (j, k) The graphs show the ratio of the number of ciliated cells
TAZ cKO mouse renal tissues. (e) Fluorescence staining of TAZ with tubule- to the number of DAPI-stained nuclei per image and lengths of primary
specific markers in WT. Synaptopodin, renal podocytes; Calbindin, distal cilia. (l) The graphs show the proportions of cilia lengths in three ranges:
convoluted tubule; DBA, collecting duct. The magnified images show the <2.5 μm, 2.5 - 5 μm, and >5 μm.
cytosolic/nuclear localization of TAZ in each renal tubule.
Summary & Discussion
Fig. 2. Changes in TAZ
localization during primary Our results suggested that TAZ may regulate primary cilia in cooperation
cilia formation. with NPHP in the glomerulus. The mechanism by which TAZ regulates
(a-d) Observations of TAZ cilia formation independent of YAP needs to be further studied, and the
localization in NIH3T3 cells possibility that TAZ deficiency-induced glomerular cysts may regulate
upon serum starvation. (a) cyst formation through ciliary recovery also needs to be discussed.
Acetylated α-tubulin-labeled There is a possibility that NPHP restores glomerular cyst formation in
primary ciliary axoneme. (b) TAZ cKO mice, which could be investigated if experiments are
Comparison of YAP/TAZ protein performed to determine the relationship among TAZ, NPHP and cyst
expression under serum- formation in the glomerulus.
starvation. (c, d) The graph
shows the fluorescence intensity of TAZ around primary cilia and ratio of Jong Hoon Park, Ph.D. Professor, Department of Biological Science Director,
the nuclear TAZ fluorescence intensity to the cytosolic TAZ fluorescence Post Genome Research Center & Research Certer for Molecular Medicine,
Sookmyung Women's University, 100 Changpa-ro 47-gil, Yongsan-gu, Seoul
intensity at 0, 6, 12, and 24 h after serum withdrawal. http://parkjh.sookmyung.ac.kr/ E-mail: parkjh@sookmyung.ac.kr
Tel: + 82 2 710 9414 CP: 010-8817-4711