Fam20C‐mediated phosphorylation of osteopontin is critical for its secretion but dispensable for its action as a cytokine in the activation of hepatic stellate cells in liver fibrogenesis

Abstract

Amedeo Carraro and Anna Maria Brunati are co-senior authors.Abbreviations: BDL, bile duct ligation; CK1, casein kinase1; ECM, extracellular matrix; ER, endoplasmic reticulum; Fam20A, family with sequence similarity 20, member A; Fam20C, family with sequence similarity 20, member C; F/P, FBS combined with PDGF; FBS, fetal bovine serum; GRP94, glucose related protein 94; GRP78/BiP, glucose related protein78; HSCs, hepatic stellate cells; OPN, osteopontin; PDI, protein disulfide isomerase; PDGF, platelet-derived growth factor; PTMs, posttranslational modifications; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; RGD, Arg-Gly-Asp; SIBLING, small-integrin-binding ligand N-linked glycoprotein; Wb, Western blot; α-SMA, alpha-smooth muscle actin. AbstractOsteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G-CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500-kDa multiprotein complex, as compared with the negligible activity in livers of sham-operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen-I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis. K E Y W O R D Sbile duct ligation, Golgi apparatus, hepatic stellate cells, osteopontin, phosphorylation

Prevention of Arterial Elastocalcinosis: Differential Roles of the Conserved Glutamic Acid and Serine Residues of Matrix Gla Protein

“…The phosphorylation of serine residues arranged in a similar motif in osteopontin has been linked to its secretion. 35 Considering this, we first examined whether in cultured cells, the mutated protein can be expressed, localized, and secreted in a comparable manner to that of the native protein. Because of the unavailability of a suitable antibody to detect mouse MGP in immunodetection techniques, we tagged both wild type and mutated MGP with the FLAG tag.…”

Comprehensive analysis of an endoplasmic reticulum stress-related gene prediction model and immune infiltration in idiopathic pulmonary fibrosis

“…Cytidine deaminase (CDA) is an enzyme that plays a crucial role in the pyrimidine salvage pathway ( 71 ). Family with sequence similarity 20 member C (FAM20C), previously known as Golgi casein kinase (G-CK), is a protein specifically localized in the Golgi apparatus ( 72 ). NELL2 is a glycoprotein involved in the development of neural cells and guidance of axons, including their repulsion ( 73 ).…”

Regulation of secretory pathway kinase or kinase-like proteins in human cancers

“…FAM20C promotes the phosphorylation of secretory proteins under diverse physiological and pathological conditions. These proteins participate in the interaction between the ER and the GA. FAM20C is involved in the supramolecular mechanism directing protein sorting and promoting the appropriate molecule conformation outside the cell ( 172 ). It is suggested that the pathological inhibition of FAM20C can inhibit abnormal protein sorting.…”