Page 57 - F. Cell biology

Prominin-1 Radixin Axis controls the trafficking of various bile canaliculi
proteins by regulating PKA activity in hepatocytes
Young Jae Kwon , Hyun Lee and Young-Gyu Ko *
#
#
Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
BACKGROUND AIM

Prominin-1(Prom-1, also known as CD133), pentaspan transmembrane Find function of prom1 in liver. In detail, we try to
glycoprotein, is specifically localized plasma membrane protrusions. In the liver,
Prom-1 is expressed in several cell types, but its physiological role is almost elucidate correlation between bile canaliculus
unknown. We observed that Prom1 is primarily localized at the canalicular structure and prom1 in lithogenic diet model by using
membrane of hepatocytes. When we investigated phenotype during lithogenic diet Prom1 wild type, knock-out mouse. Moreover,
model in Prom1+/+ and Prom1-/- mice, Prom1 deficiency showed a more
aggravated phenotype by lithogenic-induced cholestatic liver injury. investigate the mechanism of prom1-PKA signaling.

METHODS

Animal models. Prom1 knockout mice were purchased from The Jackson Laboratory (Stock NO. 017743, Bar Harbor, ME, USA). The Prom1-/- mice were backcrossed with C57BL/6N mice for
five generations. All animal studies were conducted with the approval of the Korea University Institutional Animal Care and Use Committee and the Korean Animal Protection Law (KUIACUC-
2018-6 and -2019-0111).
Diet model. To induce lithogenic in mouse, mice are fed the cocoa butter diet(Envigo, TD.88051) during 2weeks.
Immunohistochemistry. Each paraformaldehyde-fixed samples were either embedded in paraffin or frozen in OCT compound and cut into 5um-thick sections. Tissue samples were then
stained according to standard protocol, and the images were captured on light microscope (Leica).
Quantitative real-time PCR. RNA (4 μg) was reverse transcribed to cDNAs using random hexamer primers, oligo dT and Reverse Transcription Master Premix (ELPIS Biotech, Daejeon,
Korea). Quantitative real-time PCR analyses were performed using the cDNAs from the reverse transcription reactions and gene-specific oligonucleotides (Appendix Table S2) in the presence of
TOPreal qPCR 2X premix (Enzynomics, Daejeon, Korea).
Statistical analysis. Values are presented as means ± SEM. A two-tailed Student’s t-test was used to calculate the P values

RESULTS

In the liver, Prom-1 is expressed in several cell Figure 1 B Prom +/+ Prom -/- Figure 2
types, but its physiological role is almost unknown. A 100 Prom1 β-gal SD LD A NCD Litho
Here, We observed that Prom1 is primarily localized 80 ***
at the canalicular membrane of hepatocytes. When 60 Prom1/Dapi Prom1 +/+
we investigated phenotype during lithogenic diet Relative mRNA expression 40 C Prom +/+ Prom -/- Zo-1/ CK19/ GS/ DAPI
model in Prom1+/+ and Prom1-/- mice, Prom1 20 SD LD SD LD
deficiency showed a more aggravated phenotype by 0 Prom1 -/-
KO
WT
KO
WT
lithogenic-induced cholestatic liver injury. CON CON Litho Litho
Furthermore, when the canaliculi membrane Figure 3 A B
transporters were detected with Immunohistochem.
in mice undergoing lithogenic diet, the canalicular
localization of proteins was significantly reduced in
Prom1-/- mice. Because some canaliculi membrane
transporters are recruited from intracellular pools to
the bile canaliculi by PKA(Protein kinase A)
signaling, we investigated the relevance of this
machinery in lithogenic diet induced events.

Figure1. Lithogenic diet induces prom1 upregulation in liver. (A) Relative expression of Prom1 were determined by RT-qPCR (n=6) (B) Prom1 is detected by
immunohistochemistry. (C) Prom1 is overexpressed when fed a lithogenic diet.
Figure 2. Bile canaliculus structure is aggravated prom1 KO mouse in lithogenic diet model. (A) Bile canaliculus is collapsed in Prom1 KO which is fed lithogenic diet.
Figure 3. PKA signaling is more activated in prom1 WT mouse than prom1 KO. (A) p-PKA substrate is upregulated in prom1 wild type lithogenic diet model. Each of
the different transporter proteins has its own expression patterns. (B) Bile canaliculus structure is destroyed in Prom1 KO lithogenic diet model. Actin and ZO-1 are
used as canaliculus marker. Mrp2 and ABCC1 are common membrane transporter proteins.

CONCLUSION REFERENCES

As a result, The prom1 deficiency 1. Eun-Ji Lee et al. Proteasome inhibition protects against
modulation
gallstone
formation
of
through
diet-induced
inhibited cAMP dependent protein cholesterol and bile acid homeostasis. International Journal of
kinase A(PKA) activation and migration Molecular Medicine. 41, 1715-1723 (2017)
of various transporters to the 2. Helen H. Wang et al. Evidence that the adenosine
triphosphate‐binding cassette G5/G8‐independent pathway
canalicular domain under pathological plays a determinant role in cholesterol gallstone formation in
conditions. Based on these results, we mice. Hepatology. 64, 853-864 (2016) Contact information
conclude that Prom1 regulates the 3. Liqin Zhu et al. Prominin 1 marks intestinal stem cells that TEL : 02-3290-3961
are susceptible to neoplastic transformation. Nature. 457, 603–
transport of various membrane 607 (2009)
transporters to canaliculi by PKA 4. Effect of Ezetimibe on the Prevention and Dissolution of E-mail : kyj9302@korea.ac.kr
Cholesterol Gallstones. GASTROENTEROLOGY. 134, 2101–
signaling in liver injury condition. 2110 (2008)