Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis

Abstract

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

The UBR box E3 ligases Poe and Hyd are required for efficient Pericentrin degradation

“…Interestingly, Rad6 (E2) is known to function with Poe (E3) in its N-end rule regulation of proteins involved in vesicular trafficking and the lysosomal pathway (Hong, 2015). Hyd (E3) has been studied in context of the N-end rule in regulating Hedgehog signaling, oogenesis, and immune response in Drosophila (Cammarata-Mouchtouris, 2020;Dorogova, 2020;Moncrieff, 2015), while its human ortholog UBR5 was studied in ovarian cancer signaling, tissue homeostasis, and miRNA mediated translational control (Cho, 2017;Hughes, 2021;Mellis, 2021;Tasaki, 2012). Finally, while UbcD1 (E2) has not been linked to the N-end rule, it does show high similarity to UBC4 (Treier, 1992), which is an E2 known to function with 13 Hyd (E3) /UBR5 in yeast (Stoll, 2011).…”

The role of E3 ubiquitin ligases in bone homeostasis and related diseases

“…Moreover, another E3 ligase ubiquitin protein ligase E3 component n -recognin 5 determines the releasing of SMO from Patched-1 receptors to maintain cartilage homeostasis and suppresses metaplasia. Ubiquitin protein ligase E3 component n -recognin 5 loss of function in mice induces the serious degradation of articular cartilage, aberrant sesamoid bones and extensive heterotopic tissue metaplasia 119 .…”

Integral Membrane Protein 2A Is a Negative Regulator of Canonical and Non-Canonical Hedgehog Signalling

“…In preparation for transfection, cells were seeded in antibiotic-free media into culture dishes of different areas and transfected with Lipofectamine 2000 when they reached 80-90% confluency, following the manufacturer's instructions. To generate shRNA stable cells, cells were transduced with lentiviral particles, encoding scrambled or ITM2A targeting shRNA sequences (Table 1), produced in HEK293T cells as previously described [21].…”