Regulation of Embryonic Stem Cell Pluripotency by Heat Shock

Protein 90 ERIC BRADLEY, ERHARD BIEBERICH, NAHID F. MIVECHI, DANTERA

TANGPISUTHIPONGSA, GUANGHU WANG

STEM CELLS 2012

Speaker: Abelrasoul, Mahmoud Shaaban

2013-1-08

Origin of embryonic stem cells Sperm & Ovum

fert i l izat ion

zygote

Equal Divisions

two, four, eight… blastomeres

Reararrangements

Blastocyst

Inner cell mass

Pluripotent ESCs

Why embryonic stem cells?

• ESCs have self-renewal and pluripotency.

• Undifferentiated ESCs can form any adult cell.

Factors maintaining ESCs pluripotency

• Nanog• Oct 4• Sox 2• LIF• c-Myc• Klf4• Stat3 …..

Molecular Chaperone• Proteins that assist the non-covalent folding or unfolding of

other macromolecules.

• Prevent newly synthesized ploypeptides from aggregation.

• Alzheimer's disease, amyloid fibrils.

Nature 416, 483-484 (4 April 2002) | doi:10.1038/416483a 

Heat shock protein 90(Hsp90)

• Important molecular chaperon participates in stabilizing and activating more than 100 proteins referred to as Hsp90 ‘‘client proteins’’.

• Regulate proteostasis (eukaryotic protein homeostasis)

• Hsp90β: highly expressed.• Hsp90α: stress-inducible.

L. Neckers, CCR

Chaperone Machinery

• Hsp90, Hsp70, and other co-chaperones, such as Hop, form a dynamic complex known as the Hsp90 chaperone machinery.

• Cancer cells use this machinery to prevent degradation and misfolding of mutated and overexpressed oncoproteins .

• So, Hsp90 is an oncogene tolerance and cancer cell survival.

Leukemia inhibitory factor(LIF)• LIF is an interleukin 6 class cytokine

affects cell growth by inhibiting differentiation.

• When LIF levels drop, the cells differentiate.

• LIF can maintain pluripotency of ESCs by the phosphorylation and subsequent nuclear translocation of Stat3 via an Interleukin-6 pathway.

•Stat3 is physically associated with Hsp90, Song H et al 2004.•LIF promotes Hsp90 association with Stat3 in mouse ESCs, Setati MM et al 2010.

Mol Cancer Ther 2007;6:2386-2390

The purpose of the work

• To give an evidence that Hsp90 is required for pluripotency in mouse ESCs by using multiple approaches.

• To show that Oct4 and Nanog are potential Hsp90 client proteins.

Materials and Methods

• Hsp90 inhibited by 17-AAG in J1 mouse ESCs.• To determine ESCs pluripotency after

treatment we examined:1. Morphology.2. AP staining. 3. Teratoma formation.4. Pluripotency protein markers (Oct4, Nanog,

and phosphorylated Stat3).

Results and Discussion

• Figure 1. A. showing dome-shaped control ECSs and extended flattened 17-AAG treated ESCs.

• Dose: 250 µM 17-AAG for 48 hours. Scale bar= 30 µm.

Morphology changed

•Alkaline phosphatase (AP) staining of the mouse ESC colonies.

•Red color refers to AP +ve stained undifferentiated ESCs (pluripotent).ECSs loosed pluripotency by Hsp90

inhibition

• Teratoma formation 17-AAG treated or untreated cells were injected intracranially into P3 mouse brains. Mice were sacrificed, and relative teratoma weight was measured 4 weeks later.

Teratoma size decreased by more than 50% by 17-

AAG treatment

• Western blot analysis of pluripotency factors. pStat3, tStat3, Nanog, and Oct4 were detected using 250 nM 17-AAG-treated mouse ESC samples

levels of the pluripotency factors Oct4, Nanog, and pStat3 were

greatly diminished in the 17-AAG-

treated samples

Similar phenotypes were observed when the ESCs were treated by another Hsp90 inhibitor GA

Hsp90 levels during differentiation into embryoid bodies (EBs).

• Figure S2 Hsp90 proteins were down-regulated during ES differentiation into EBs. Cell lysates were collected at stages indicated and Westernblot analysis performed for Hsp90α and Hsp90β. ES, feeder free ESCs; EB1-1, floating EBs 24 hours after plating; EB1-2, floating EBs 48 hours after plating; EB2-1, attached EBs 24 hours after attached to the tissue culture dishes; EB2-2, attached EBs 48 hours after attached to the tissue culture dishes.

Hsp90 Inhibition by MicroRNA

• Western blot analysis showed the Characterization of miRNA against Hsp90α and Hsp90β in NIH3T3 cells. miRNAs A2139 and A2161 against Hsp90α, and B1201, B1626 against Hsp90β were transfected into NIH3T3 cells.

Hsp90 Inhibition by MicroRNA

• When miRNA A2139 and B1201 were electroporated into mouse ESCs, we found that these miRNAs effectively reduced the protein levels of Hsp90α and Hsp90β, and a mixture of A2139 and B1201 reduced both.

Oct4 and Nanog reduced by Hsp90

miRNAs

Hsp90 Inhibition by MicroRNA

• Hsp90 miRNAs significantly reduced the number of AP posi-tive colonies, with the double transfection of both Hsp90α and Hsp90β miRNA being the most potent.

• Rescue assay, Overexpression of Hsp90β by a lentiviral vector partially restored the loss of the ESC pluripotency as determined by AP staining and western blot analysis.

Is Hsp90 Associated with Oct4 and Nanog ?and Can Protects them from degradation through Ubiquitin proteasomal degradation pathway ?????

• both Hsp90α and Hsp90 β pulled down endogenous Oct4 from ESC lysates. In the reciprocal immunoprecipation reaction, Oct4 antibody pulled down both endogenous Hsp90α and Hsp90β.

• Similarly, endogenous Nanog and Hsp90 were found to be pulled down by each other.

• Inhibition of the ubiquitin proteasomal degradation pathway by MG132 in 17-AAG-treated ESCs.

• MG132 treatment prevented the Nanog and Oct4 downregulation mediated by Hsp90 inhibition.

Oct4 and Nanog are potential novel Hsp90 client proteins, through

which Hsp90 participates in the maintenance of ESC pluripotency.

Hsp90 Inhibition Reduced Oct4 mRNA Level in Both Mouse and Human ESCs

• Oct4 mRNA level was reduced by 17-AAG in mouse ESCs in a dose-dependent manner, while Nanog, Sox2, c-Myc, and Klf-4 mRNA levels remained intact.

• Quantitative real-time PCR indicated that Hsp90 inhibition by 17-AAG reduced the Oct4 mRNA level in mouse and human ESCs.

• These data indicate that Hsp90 regulates Oct4 mRNA transcription, maturation, or stability in ESCs.

Mouse ESCs Human

ESCs

• Oct4 mRNA level was downregulated by 17-AAG in EBs.• The primitive ectoderm marker Fgf5 was reduced, while the

primitive endoderm marker GATA4 remained unchanged.

Hsp90 inhibition in Dose-dependent manner

• (A): Feeder-free mouse ESCs were treated for 48 hours with 17-AAG of concentrations indicated. Cell lysates were collected, and Western blot analysis was performed for proteins indicated.

HOP co-chaperone is increased as a potential

compensatory response.

• Figure S5. Hsp90 inhibition induced apoptosis.

Was the pluripotency loss mediated by Hsp90 inhibit ion as a result of increased cell death ?

No, it is not a secondary effect of

apoptosis.

• Figure 6. Heat shock protein 90 (Hsp90) inhibition increases markers for mesoderm differentiation.

• AAG, allylamino-17-demethoxygeldanamycin; AFP, a-fetoprotein; Con, vehicle treated; Hoe, nuclei staining with Hoechst; SM, smooth muscle.

Conclusions

• Hsp90 can maintain the ESCs pluripotency.

• Pluripotency loss is prior to apoptosis activation. Thus, Hsp90 is required for cell survival too.

• Oct 4 and Nonog are potential Hsp90 client proteins.

• After Hsp90 inhibition by 17-AAG, the ESCs increased the expression of mesoderm markers desmin and protein T, implying that they might preferentially differentiate into mesoderm lineages.

• Hsp90 reduced the mRNA level of Oct4. This may resulted from ESCs differentiation into mesoderm lineages.