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Identification and characterization of endothelin receptors on rat osteoblastic osteosarcoma cells: down-regulation

P Nambi1, H L Wu, D Lipshutz

  • 1Department of Renal Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.

Molecular Pharmacology
|February 1, 1995
PubMed

Insights

This study found that rat osteosarcoma cells have functional endothelin receptors (ETA and ETB). 1,25-dihydroxy-vitamin D3 down-regulates these receptors, particularly ETA, impacting osteocalcin expression.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Endocrinology

Background:

  • Endothelins (ETs) are peptides involved in various biological processes.
  • A role for ETs in bone remodeling is suggested, necessitating further investigation.
  • The rat osteosarcoma cell line ROS 17/2.8 is a model for studying bone cells.

Purpose of the Study:

  • To characterize endothelin receptors (ETA and ETB) and their signaling pathways in ROS 17/2.8 cells.
  • To investigate the regulation of these receptors by 1,25-dihydroxy-vitamin D3.
  • To determine the role of ETA receptors in osteocalcin expression.

Main Methods:

  • Radioligand binding assays using 125I-ET-1 and 125I-IRL-1620 to quantify ETA and ETB receptors.
  • Measurement of inositol phosphate accumulation and intracellular Ca2+ release to assess receptor signaling.
  • Western blot analysis to detect osteocalcin protein expression.
  • Quantitative analysis of ETA and ETB receptor mRNA levels.

Main Results:

  • ROS 17/2.8 cells express high-affinity ETA and ETB receptors (3:1 ratio).
  • ET-1 and sarafotoxin 6c activate phospholipase C, leading to increased inositol phosphate and Ca2+ release.
  • ET-1, acting via ETA receptors, induces osteocalcin protein expression.
  • 1,25-dihydroxy-vitamin D3 significantly down-regulates both ETA and ETB receptor binding, primarily by reducing receptor number.
  • 1,25-dihydroxy-vitamin D3 decreases ETA receptor mRNA levels but has minimal effect on ETB mRNA levels.

Conclusions:

  • ROS osteoblasts possess functional ETA and ETB receptors coupled to phospholipase C.
  • ETA receptor activation is crucial for ET-1-induced osteocalcin expression.
  • 1,25-dihydroxy-vitamin D3 down-regulates endothelin receptors at both protein and mRNA levels, with a more pronounced effect on ETA receptors.

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