Integrated quantitative proteomics and phosphoproteomics analysis reveals USP46-POU4F1-HPSE signaling axis in the pathogenesis of Hirschsprung disease

Abstract

Hirschsprung’s disease (HSCR) is a congenital disorder characterized by the absence of enteric ganglion cells in the distal colon, resulting in functional intestinal obstruction. While genetic mutations and microenvironmental imbalances have been implicated in HSCR, the underlying molecular mechanisms are not fully understood. This study uses integrated quantitative proteomics and phosphoproteomics analyses to characterize the differential protein profiles and phosphorylation modifications associated with HSCR. These findings reveal significant dysregulation of the extracellular matrix (ECM) remodelling pathway, suggesting its potential involvement in HSCR pathogenesis. Notably, the deubiquitinating enzyme USP46 is found to be significantly reduced in the aganglionic segments of HSCR patients. Through IP-MS, GST pull-down, and co-immunoprecipitation assays, it is demonstrated that USP46 interacts with the transcription factor POU4F1. Mechanistically, USP46 stabilizes POU4F1 via deubiquitination, increasing its binding to the heparanase (HPSE) promoter and increasing HPSE expression, which in turn promotes ECM remodelling and neural cell migration. The role of the USP46-POU4F1-HPSE signaling axis in HSCR pathogenesis is confirmed via chromatin immunoprecipitation-qPCR, luciferase reporter assays, and transwell migration assays. This study elucidates a novel regulatory mechanism linking USP46-mediated protein stabilization to ECM dynamics and neural cell migration, offering new insights into HSCR pathogenesis and potential therapeutic targets.

1. Introduction

Hirschsprung’s disease (HSCR) is a congenital disorder characterized by the absence of ganglion cells in the distal intestine, resulting in functional bowel obstruction, chronic constipation, and Hirschsprung-associated enterocolitis (HAEC) [1]. It occurs in approximately 1 out of every 5000 live births, with males being four times more likely to be affected than females are, suggesting possible sex-linked genetic factors [2]. Even with surgical intervention through procedures such as Swenson, Soave, or Duhamel pull-through, up to half of patients may experience post-surgical issues such as ongoing constipation, enterocolitis, and fecal incontinence [3,4]. This highlights the need for a deeper understanding of its pathophysiology, which involves complex interactions of various cellular and molecular components within the colonic segments.

HSCR is a multifactorial disorder with significant genetic components. The proper function of the enteric nervous system (ENS) depends on the interactions between neurons and glial cells. Originating from neural crest cells (NCCs), which migrate and differentiate into enteric ganglia during embryogenesis, ENS development can be disrupted by genetic mutations, including those in the RET proto-oncogene, EDNRB, GDNF, and SOX10 [5,6]. These mutations disrupt NCC migration, proliferation, or differentiation, leading to HSCR. Recent studies have identified new candidate genes, such as ATP7A, SREBF1, ABCD1, and PIAS2, expanding the known genetic landscape of HSCR [6]. The RET/GDNF signaling pathway is crucial for enteric neural crest cell (ENCC) migration, survival, and differentiation [7]. However, known genetic mutations account for only approximately 20% of sporadic HSCR cases, indicating that additional regulatory mechanisms are involved [8]. Even among patients with HSCR-associated mutations, high phenotypic variability suggests that non-genetic factors such as epigenetic regulation and posttranslational modifications contribute to the disease [9,10]. These findings underscore the need to investigate posttranslational regulators involved in ENS development.

The extracellular matrix (ECM) is a critical regulator of ENS progenitor migration, providing both structural support and biochemical signaling [11,12]. In the context of ENS development, several ECM components play key roles: collagen VI (COL6A3) and fibronectin (FN1) regulate the adhesion and differentiation of enteric neural crest cells (ENCCs) [13], and heparanase (HPSE), a key ECM-degrading enzyme, modulates ECM turnover and enhances neural crest cell migration [14]. In HSCR, dysregulated ECM remodelling results in excessive ECM deposition. This accumulation impairs neural crest cell migration and contributes to the failure of ENS development [15,16]. Our previous research demonstrated altered expression levels of collagens I, III, and IV in the colon segments of HSCR patients, changes that may compromise the structural integrity and function of the ENS [17]. However, the molecular mechanisms that connect ECM regulation with HSCR pathogenesis still require further investigation.