Regulation of thrombopoietin receptor expression and function

Of the many cells in the body, the hematopoietic cells are among those with the highest rate of self-renewal and turnover. The production and destruction of these cells are tightly controlled by a number of hematopoietic growth factors, in particular by members of the family of helical cytokines. Studying the thrombopoietin receptor, I focused on two aspects of cytokine receptor signaling: attenuation of signaling by receptor isoforms and the biological function of cytokine receptor target genes. Cytokine receptor signaling has profound effects on cell survival, proliferation and differentiation. It is therefore not surprising that components of the signaling cascade are tightly regulated at the level of expression. An important mechanism for controlling gene expression is alternative splicing. Alternate isoforms have been identified for many cytokine receptors and a regulatory function and/or altered expression in disease have been described for some of these isoforms The cytokine thrombopoietin (TPO) and its cognate receptor c-mpl are the primary regulators of platelet production and also play an important role in hematopoietic stem cell biology. Several isoforms of unknown function exist for both mouse and human mpl and it is possible that they play an important role in modulating mpl signaling. In my thesis work, I have analyzed the function of a truncated receptor isoform (mpl-tr) which is the only alternate mpl isoform conserved between mouse and humans. Although mpl-tr lacks a transmembrane domain, classifying it as a ‘secreted’ or ‘soluble receptor’, it is retained intracellularly. My results provide evidence that mpl-tr acts as a dominant-negative variant of mpl for both proliferation and survival. I also demonstrate that mpl-tr mediates protein degradation of the full-length receptor by a cathepsin-like cysteine protease activity. Due to a shift of the reading frame at a splice acceptor site, the C-terminus of mpl-tr consists of a peptide of unique sequence, 30 amino acids in length. I show that this peptide sequence is essential for the inhibition of TPO-dependent proliferation and for mpl protein degradation mediated by mpl-tr. Together, these data suggest a new paradigm for the regulation of cytokine receptor expression and function
through a proteolytic process directed by a truncated isoform of the same
receptor. To test for the in vivo function of alternative mpl isoforms, a c-mpl cDNA was
expressed as a transgene in mpl knockout mice. These mice express mpl fulllength
as the only mpl isoform and develop severe thrombocytosis with
platelet numbers, elevated about five times higher than normal. The
reintroduction of the endogenous mpl allele restores normal platelet counts
and I attribute this to the in vivo effect of dominant-negative mpl isoforms. A
mpl knock-in allele, which does not express mpl-tr but still expresses the
second known alternate variant of murine mpl, mpl-II, normalizes platelet
numbers, similar to the endogenous mpl allele. This result demonstrates that
the absence of mpl-tr is not sufficient to cause thrombocytosis. I propose that
mpl-II is an additional dominant-negative mpl isoform and attenuates the
expansion of the megakarocytic lineage in vivo. In summary, these results
impressively demonstrate the importance of alternate cytokine receptor
isoforms in vivo and emphasize the need to study the function of the many
uncharacterized cytokine receptor isoforms.
In a second project, I studied the role of mpl signaling in regulating the
expression of a gene with a potential role in cell differentiation and
proliferation. The diversification of cell types is controlled through the use of
both lineage-restricted and more widely expressed transcriptional regulators
and the combinatorial actions of these regulators specify gene expression.
The differentiation of megakaryocyte precursors is dependent on the proper
function of the GATA-1 transcription factor. Mice lacking GATA-1 selectively in
megakaryocytes have dramatically fewer platelets but more megakaryocytes,
altered platelet size and shape and prolonged bleeding times. Further, GATA-
1-null megakaryocytes hyperproliferate in vitro, suggesting that GATA-1 is
both a differentiation factor and negative regulator of megakaryocyte cell
proliferation. However, GATA-1 regulated genes which are responsible for this
growth inhibition are presently unknown. In this thesis work, I describe a novel
gene, GASIP (GATA-1 regulated SIAH Interacting Protein), which is dramatically downregulated in mpl-transfected hematopoietic cell lines,
identifying mpl as a negative regulator of GASIP expression. The presence of
juxtaposed GATA and Ets-binding cis-elements in the GASIP promoter are
typical for a megakaryocytic gene. I found that GASIP expression in platelets
is indeed robust and correlates with mRNA levels of GATA-1, but not GATA-2
or –3, identifying GATA-1 as a positive regulator of GASIP expression. The
finding that mpl and GATA have opposite effects on both proliferation and on
GASIP expression, make GASIP a candidate GATA-1 target gene involved in
growth inhibition. To investigate the potential role of GASIP in growth
regulation, I screened for potential protein binding partners. Interestingly, I
identified the p53-inducible tumor suppressor seven in absentia homologe
(SIAH) as a GASIP interacting protein. I speculate that GASIP may contribute
to the anti-proliferative effect mediated by SIAH.