1) What two fundamental differences between the retinoic acid (hormone) signaling pathway and other signaling pathways exists?
Retinoic acid
Other pathways
Retinoic Acid signaling works with intracellular nuclear receptor (primarily nuclear hormone receptors (RARs or RXRs [retinoid X receptors])
other signaling pathways have cell surface receptors
RA acts as a transcription factor & directly regulates gene expression by translocating into the nucleus and binding to the RAREs (Retinoic acid response elements) that directly influence the transcription machinery and epigenetics. pathway does not require intermediate signaling molecules or extensive signal amplification steps
other signaling pathways work with signal transduction cascades with proteins / kinases phosphorylation and other mechanisms
Retinoic Acid has to be produced from Vit A which again needs to be taken up in the diet so the signaling would not work without Vit A uptake
Retinoic Acid is an alcohol (small, lipophilic molecule) derived from vitamin A (retinol). can freely diffuse across cell membranes (hydrophobicity), No need for membrane-bound receptors or transport mechanisms
- other signaling molecules often large hydrophilic proteins or peptides, cannot easily cross membrane & rely on membrane-bound receptors
2) Is retinoic acid a morphogen, if yes why?
It fulfills criteria of morphogens:
formation gradient
concentration dependent manner
directly influences cell fate
diffuses through tissues, creating a gradient with high concentrations near the source of production, it is spatially and temporally regulated
By binding to RAR and their interaction with RAREs it affects the cells developemental fate and even changes their histone modification
E.g. RA is crucial for A-P patterning, limp developement, neural differentiation or organogenesis
3) How is RA metabolized and catabolized?
Catabolization:
RA bound by CYP26 -> RA hydroxylated -> water soluble -> excretion
Retinal -> storage form or directly oxidised into -> retinaldehyd -> RA
-> Binding Proteins outside and inside the cell guide the RA
Anabolization:
Retinol (Vit A) transported / diffuses into the cell through STRA6 transmembrane channel -> oxidised by alcohol dehydrogenase / retinol dehydrogenase (ADH / RDH) to Retinal (retinaldehyd) -> further oxidized to retinoic acid by retinaldehyd dehydrogenase (RALDHs)
Retinol can also first be transformed into retinyl ester by lecithin:retinol acetyltransferase (LRAT) -> storage form (in liver) and then again hydrolysed to be oxidized to RA
RA can also be produced by uptake of ß-carotine and tranforming that with a dioxygenase into retinaldehyd -> RALDH to RA
4) Explain the machanism (s) how RA activates transcription.
5) Explain the machanism (s) how RA represses transcription.
Repression:
retinoic acid receptors (RARs) and retinoid X receptors (RXRs) from heterodimers in the nucleus and in absence of RA bind to retinoic acid response elements (RAREs) located in the promoter regions of target genes where they recruit histone deacetylation complex (with NCoR/SMRT) that represses gene transcription by tightly packing the histones -> RNA poly cant bind ->transcription repression
Activation:
Upon ligand binding RAR/RXR undergoes conformational change and can then recruit Coactivation complex with histone acetylase methyltransferase … through these modifications the histone decompacts and the RNA polymerase with mediator complex and TFs can bind after Co-activator complex dissociates --> transcription activation
—> Subtype II of nuclear receptors four classifications
The four nuclear receptor subtypes:
I: NR are chaperone bound in cytoplasm and released upon ligand binding to translocate into nucleus, bind response elements as homodimers
II: NR located in nucleus, absence of ligand -> Co-repressor complex bound, presence of ligand -> Co-activator Complex bound, forms heterodimers with RXR
III: same as II but as homodimers
IV: same but as monomeres
7) What are the differences between the RA signaling reporters?
RA signaling cannot be visualized by proteins -> RA is no protein ! but an alcohol -> no antibody staining possible
FRET reporter:
RA binding domain on RARs coupled to CFP and YFP upon binding the conf. chage changes the CFP signal to YFP (energy transfer because of proximity) this reporter can distinguish between activated RARs and Inactive but is independent of target gene transcription
GFP bound to RARE:
when transcription is active GFP signal active only ! GFP very stable and big protein could cause unwanted problems —> use GFP with death domain for faster degradation by the cell
Other systems could be Gal 4 system where ligand binding protein linked with Gal 4 transcribing GFP upon activiation
Differences between reporter systems lie in Type / detection method, Promotor/response element design, sensitivity / specificity and what its actualy use / application is
6) Which signaling pathways influence the activity of the RA pathway?
Phosphorylation at serveral targets of the RA signaling pathway like the Co-repressor complex leading to its degradation or on the RARs which can have pos or neg effects depending where the P is
Many signaling pathways have this kind of influence
PKA: GPCR -> Adenylatcyclase -> cAMP -> PKA
PKC: PLC & PIP2-> IP3 & DAG -> PKC
ERK: EGFR -> RAF -> RAS -> MEK -> ERK
AKT: PI3K -> PIP2 -> PIP3 -> AKT
JNK
e.g. PKA, PKC, ERK, Akt and JNK+P38 these kinases are downstream of they cAMP pathway, G-coupled rezeptors, RTKs like for FGF signaling (MAPK/Erk), PI3K / Akt or MEKK / P38 / JNK pathway
FGF!
Insulin (PKC, RAS/MEKK/ERK)
RARs alpha at cell membrane as kinases
8) Retinoids are already used as drugs for cancer therapy. Which mechanisms of action of the RA signaling pathway are the basics?
Depending on which binding protein the RA binds to the effect can change but by downregulating FABP5 and upregulating CRABP-II the apoptotic, growth arrest and thereby anti tumor activity is mediated
low ATRA -> active RARy control stem cell maintenance
increased ATRA -> active RARa control stem cell differentiation —> mechanism that the AllTransRA induces differentiation of leukemic promyelocytes into mature granulocytes, leading to remission in APL (Acute Promyelocytic Leukemia) patients
Other then that RA signaling can have more effects for cancer therapy e.g. inhibition of angiogenesis (inhib VEGF), epigenetic modulation, upregulating differentiation of immun response cells, inhibition of Proliferation (by acting on cell cycle, like in spermatogenesis) and iduction of apoptosis
9) RA signals directly inhibt the expression of FGF8 during embryonic body axis extension. What mechanism could act here?
at somitogenesis
FGF8 gene has RARE binding site, upon RA binding RXR/RARy recruits Co-repressor complex leading to epigenetic modifications that inhibit the FGF8 transcription
10) RA has in many cancer cells antiproliferative and apoptosis inducing function. In contrast, RA promotes the emergence and growth of colon tumors. What could be the reason for this?
A reason could be the relative expression levels of FABP5 that it highly outnumbers CRABP-II —> Ligand binding protein ratio!
RA binding to FABP5 leads to proliferation and cell survival which enables even more tumor growth (oncogenic effect)
Or the RAR and RXR isoforms can vary between the cancer tissues and in some the downstream regulation promotes the tumor, same accounts for the recruitment of epicgenetic modifiers that can be repressing or activating depending on the RARE, the receptors and then again which gene is effected
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