Spop promotes skeletal development and homeostasis by positively regulating Ihh signaling

Abstract

Indian Hedgehog (Ihh) regulates chondrocyte and osteoblast differentiation through the Glioma-associated oncogene homolog (Gli) transcription factors. Previous in vitro studies suggested that Speckletype POZ protein (Spop), part of the Cullin-3 (Cul3) ubiquitin ligase complex, targets Gli2 and Gli3 for degradation and negatively regulates Hedgehog (Hh) signaling. In this study, we found defects in chondrocyte and osteoblast differentiation in Spop-null mutant mice. Strikingly, both the full-length and repressor forms of Gli3, but not Gli2, were up-regulated in Spop mutants, and Ihh target genes Patched 1 (Ptch1) and parathyroid hormone-like peptide (Pthlh) were down-regulated, indicating compromised Hh signaling. Consistent with this finding, reducing Gli3 dosage greatly rescued the Spop mutant skeletal defects. We further show that Spop directly targets the Gli3 repressor for ubiquitination and degradation. Finally, we demonstrate in a conditional mutant that loss of Spop results in brachydactyly and osteopenia, which can be rescued by reducing the dosage of Gli3. In summary, Spop is an important positive regulator of Ihh signaling and skeletal development.T he human skeleton provides essential mechanical support, mobility, and mineral storage critical for health (1). In development, most bones form through endochondral ossification in which chondrocytes in the growth plate proliferate, undergo hypertrophic differentiation, and secrete calcium-containing extracellular matrix (2). The osteoblasts in the perichondrium, a thin layer of tissue surrounding the cartilage, replace the dying chondrocytes and secrete more bone matrix. On the other hand, osteoclasts, derived from white blood cells, invade and digest bone matrix. The balance between the osteoblast and osteoclast activities allows calcium homeostatic control and bone health. In osteopenia and osteoporosis, conditions afflicting more than 10 million Americans, an abnormal decrease in osteoblast activity or increase in osteoclast activity results in the loss of bone mass (1, 3). Unfortunately, our understanding of these bone diseases has been hindered by incomplete knowledge in the molecular mechanisms underlying endochondral bone development and remodeling.Indian Hedgehog (Ihh), a member of the Hedgehog (Hh) family of signaling proteins, is essential for endochondral bone development (4). Ihh regulates gene expression through the Gli family of transcription factors, which act as both transcriptional activators and repressors (5). In the absence of Hh, efficient proteolytic processing turns Gli3 into a transcriptional repressor (Gli3R) whereas processing of Gli2 is rather inefficient (6-8). Hh inhibits Gli processing and converts the full-length Gli proteins into transcriptional activators. Both Gli2 and Gli3 play important roles in the regulation of bone formation downstream of Ihh (9-12).Ihh maintains the expression of parathyroid hormone-like peptide (Pthlh) (Mouse Genome Informatics) in the periarticular perichondrium (13), which then stimulates the prol...

Spop regulates Gli3 activity and Shh signaling in dorsoventral patterning of the mouse spinal cord

“…The other mutant allele, Spop ΔEx , bears a deletion of the 4 th and 5 th exons of Spop , which was predicted to truncate the Spop protein at residue 26 due to a frame shift. Immunoblot analyses confirmed the absence of Spop protein in embryonic day (E) 10.5 homozygous mutant embryos for both alleles (Cai and Liu, 2016). Both Spop lacZKI and Spop ΔEx homozygous mutants exhibited neonatal lethality with skeletal defects (Cai and Liu, 2016).…”

“…Immunoblot analyses confirmed the absence of Spop protein in embryonic day (E) 10.5 homozygous mutant embryos for both alleles (Cai and Liu, 2016). Both Spop lacZKI and Spop ΔEx homozygous mutants exhibited neonatal lethality with skeletal defects (Cai and Liu, 2016). A small number of mutant embryos exhibited exencephaly (n= 11/157 homozygotes and 5/407 heterozygotes, combining both alleles) and spina bifida (n= 4/157 homozygotes, combining both alleles), suggesting that Spop may play a role in the development of the nervous system.…”

A catalog of single nucleotide changes distinguishing modern humans from archaic hominins

“…RUNX2, EDAR, and GLI3(Adhikari et al 2016), NFATC1(Kim and Kim 2014), SPOP(Cai and Liu 2016), DDR2) and NELL1, possibly carrying changes in regulatory regions, while mutations in the HHMC-carrying gene encoding for the transcription factor ATRX cause facial dysmorphism(Moncini et al 2013). In addition, genes with HHMCs such as PLXNA2(Oh et al 2012), EVC2(Kwon et al 2018), MEPE(Gullard et al 2016), OMD(Tashima et al 2015), and SPAG17(Teves et al 2015) are known to affect craniofacial bone and tooth morphologies.…”

E3 Ubiquitin Ligases in Cancer and Their Pharmacological Targeting

“…Other notable SPOP substrates include the apoptotic protein Daxx [138,139], deSUMOlyase SENP7 [140], c-Myc [141], HDAC6 [142], Cdc20 [143], protooncogene DEK [144], phosphatases PTEN and Dusp7 [139], hedgehog pathway proteins Gli2 and Gli3 [145,146], and BET transcriptional coactivators BRD2-4 [147][148][149]. SPOP is also closely tied to hormone-activated pathways, as steroid receptor coactivator SRC-3 [150], androgen receptor (AR) [151], enhancer of ARmediated transcriptional activity TRIM24 [144], and estrogen receptor α (ERα) [136] are all substrates of SPOP.…”