Rapid hepatic clearance of full length CCN-2/CTGF: a putative role for LRP1-mediated endocytosis

Abstract

CCN-2 (connective tissue growth factor; CTGF) is a key factor in fibrosis. Plasma CCN-2 has biomarker potential in numerous fibrotic disorders, but it is unknown which pathophysiological factors determine plasma CCN-2 levels. The proteolytic amino-terminal fragment of CCN-2 is primarily eliminated by the kidney. Here, we investigated elimination and distribution profiles of full length CCN-2 by intravenous administration of recombinant CCN-2 to rodents. After bolus injection in mice, we observed a large initial distribution volume (454 mL/kg) and a fast initial clearance (120 mL/kg/min). Immunosorbent assay and immunostaining showed that CCN-2 distributed mainly to the liver and was taken up by hepatocytes. Steady state clearance in rats, determined by continuous infusion of CCN-2, was fast (45 mL/kg/min). Renal CCN-2 clearance, determined by arterial and renal vein sampling, accounted for only 12 % of total clearance. Co-infusion of CCN-2 with receptor-associated protein (RAP), an antagonist of LDL-receptor family proteins, showed that RAP prolonged CCN-2 half-life and completely prevented CCN-2 internalization by hepatocytes. This suggests that hepatic uptake of CCN-2 is mediated by a RAP-sensitive mechanism most likely involving LRP1, a member of the LDL-receptor family involved in hepatic clearance of various plasma proteins. Surface plasmon resonance binding studies confirmed that CCN-2 is an LRP1 ligand. Co-infusion of CCN-2 with an excess of the heparan sulphate-binding protamine lowered the large initial distribution volume of CCN-2 by 88 % and reduced interstitial staining of CCN-2, suggesting binding of CCN-2 to heparan sulphate proteoglycans (HSPGs). Protamine did not affect clearance rate, indicating that RAP-sensitive clearance of CCN-2 is HSPG independent. In conclusion, unlike its amino-terminal fragment which is cleared by the kidney, full length CCN-2 is primarily eliminated by the liver via a fast RAP-sensitive, probably LRP1-dependent pathway.

Connective tissue growth factor (CTGF) from basics to clinics

“…As a secreted soluble protein of 36-38 kD, CTGF can also be found in the extracellular body fluids including blood plasma, from where it is eliminated by glomerular filtration in the kidney, and in the liver by uptake in hepatocytes. Full length CTGF is eliminated through a rapid route, which is mediated by low-density lipoprotein receptor-related protein 1 (LRP1) binding on the hepatocytes and, to a lesser extent, (12% of total CTGF) by the kidney [53]. The C-terminal fragments of CTGF, similar to the full length CTGF, is cleared LRP1 binding on hepatocytes, which is a faster route than glomerular filtration [53].…”

“…Full length CTGF is eliminated through a rapid route, which is mediated by low-density lipoprotein receptor-related protein 1 (LRP1) binding on the hepatocytes and, to a lesser extent, (12% of total CTGF) by the kidney [53]. The C-terminal fragments of CTGF, similar to the full length CTGF, is cleared LRP1 binding on hepatocytes, which is a faster route than glomerular filtration [53]. Full length CTGF and its N-terminal and C-terminal fragments are filtered by the glomeruli and then reabsorbed completely from primary urine by the megalin-cubulin complex on proximal tubular epithelial cells.…”

CCN Family Proteins in Cancer: Insight Into Their Structures and Coordination Role in Tumor Microenvironment

“…The thrombospondin type 1 repeat (TSP-1) domain is another common domain in CCN proteins and plays strong roles in some biological functions of tumor, primarily through interactions with lipoprotein-related receptors ( Gerritsen et al, 2016 ), vascular endothelial growth factor (VEGF) ( Tsai et al, 2017 ), diverse integrins ( Alday-Parejo et al, 2019 ), and heparan sulfate proteoglycans (HSPGs) ( Neubauer et al, 2017 ). As the TSP-1 domain is conserved across CCN family members, this suggests that all CCN family members modulate cell adhesion, maintains ECM composition, and participates in regulating tumor signaling ( Jayakumar et al, 2017 ).…”

Low Density Receptor-Related Protein 1 Interactions With the Extracellular Matrix: More Than Meets the Eye

“…CTGF accumulation is traced to the missing Lrp1-mediated clearance of CTGF ( Segarini et al, 2001 ). Lrp1-mediated endocytosis of CTGF occurs tissue-independently as it has also been shown to take place in the liver ( Gao and Brigstock, 2003 ; Gerritsen et al, 2016 ). The disrupted elastin fiber architecture and ECM disorganization upon Lrp1 deletion from smooth muscle cells was found by Muratoglu et al (2013) to increase protein levels of MMP-2, MMP-9, MT1-MMP, and serine protease high-temperature requirement factor A1 (HtrA1).…”