# The regulation of thrombopoietin levels

> **NIH NIH R01** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2022 · $539,293

## Abstract

Project Summary
Thrombopoietin (TPO), a plasma glycoprotein made in hepatocytes, binds to its cell surface receptor MPL
expressed on megakaryocytes and megakaryocyte progenitors, promoting cell proliferation and maturation. In
addition to its role in megakaryocyte development and platelet production, TPO also plays a critical role in
hematopoietic stem cell survival and maintenance. In humans, gain-of-function mutations in TPO result in
autosomal dominant thrombocytosis (high platelet counts), while loss-of-function mutations in TPO (or in its
receptor MPL) result in congenital amegakaryocytic thrombocytopenia, a disease characterized by low platelet
counts and absence of bone marrow megakaryocytes at birth with subsequent bone marrow aplasia and
failure. The transcriptional regulation and plasma clearance of TPO have been well studied. In contrast, the
mechanism by which TPO is secreted from hepatocytes and by which TPO is degraded intracellularly remain
unknown. We have shown in preliminary results that mice deficient in LMAN1 exhibit thrombocytopenia and
that the thrombocytopenia is recapitulated in mice with hepatocyte-specific LMAN1 deletion but not in mice
with hematopoietic-specific LMAN1 deletion. We have additionally shown that the plasma TPO level is low in
LMAN1 deficient mice (~50% of normal). Taken together with LMAN1’s known function as an endoplasmic
reticulum cargo receptor, these results strongly suggest that the thrombocytopenia observed in LMAN1
deficient mice is due to impaired secretion of TPO from hepatocytes. Consistent with these findings in mice, we
have shown that LMAN1 deficiency results in intracellular accumulation of TPO in human cells. Therefore, the
role of LMAN1 in regulating TPO appears to be conserved in mice and humans. In this proposal, we aim to
define the role of LMAN1 in regulating the plasma TPO levels in mice and to dissect the mechanism by which
LMAN1 regulates TPO secretion, both under a normal physiological state and in the setting of enhanced TPO
production. In additional preliminary results, we have performed an unbiased genome-scale CRISPR knock-out
screen to identify novel regulators of the intracellular TPO level. This screen, performed in biological triplicates,
followed by validation experiments, demonstrated that deletion of UBE3C results in intracellular accumulation
of TPO but no intracellular accumulation of any of the 3 control secretory proteins tested. UBE3C is an E3
ubiquitin ligase, suggesting that TPO is a substrate for UBE3C and that in the absence of UBE3C, TPO
ubiquitination is impaired, resulting in reduced degradation. Therefore, an additional aim of this proposal is to
define the role of UBE3C in regulating the intracellular TPO level, both under steady state TPO production and
in a state of high TPO production. This proposal has important implications for understanding the mechanisms
of TPO regulation at the level of its intracellular trafficking/secretion and at the level of it...

## Key facts

- **NIH application ID:** 10457889
- **Project number:** 5R01HL157062-02
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Rami Khoriaty
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $539,293
- **Award type:** 5
- **Project period:** 2021-08-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10457889

## Citation

> US National Institutes of Health, RePORTER application 10457889, The regulation of thrombopoietin levels (5R01HL157062-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10457889. Licensed CC0.

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