On the transforming growth factor beta-like activity of synthetic polypeptides comprising the amino-terminal sequence of human parathyroid hormone-related peptide.
Purified native forms of human parathyroid hormone-related peptide (PTHrp) have recently been reported to display biological activities characteristic of transforming growth factor beta (TGF-beta). The TGF-beta-like property of PTHrp may reside within the amino N-terminal PTH-receptor binding region of the polypeptide, since a synthetic analog corresponding to amino acids 1-36 of human PTHrp is as active as purified native PTHrp in bioassays specific to TGF-beta. Complete lack of structural similarity between PTHrp and TGF-beta prompted us to address the question whether copresence of the TGF-beta-like and PTH-like biological activities in the N-terminal sequence of the PTHrp molecule is a general phenomenon observable with different N-terminal PTHrp peptides of varying amino acid chain length in a variety of target cells that respond in defined ways to TGF-beta in vitro. Two forms of synthetic N-terminal human PTHrp, PTHrp-(1-34) and [Tyr40]PTHrp-(1-40), which are fully active in conventional assays for PTH/PTHrp, were tested for effects in three in vitro bioassay systems for TGF-beta: 1) stimulation, and 2) inhibition, respectively, of epidermal growth factor-dependent soft-agar colony formation of either normal rat kidney-derived fibroblasts (NRK 49F) or human lung carcinoma cells (A549); and 3) biosynthesis of metabolically labeled fibronectin in both NRK 49F cells and clonal osteoblastic rat osteosarcoma cells (ROS 17/2.8). Human TGF-beta over the dose range of 2.5-80 pM significantly stimulated or inhibited soft-agar colony formation of either NRK 49F or A549 cells, respectively, and caused a severalfold increase in biosynthetically labeled [35S]fibronectin in NRK 49F and ROS 17/2.8 cells. In contrast, none of PTHrp-(1-34), [Tyr40]PTHrp-(1-40), and synthetic human PTH-(1-34), each tested at 0.1-10 nM, displayed detectable biological activity in any of the three assay systems. In addition, covalent cross-linking of intact NRK 49F and ROS 17/2.8 cells with either [125I]TGF-beta or 125I-[Tyr40] PTHrp-(1-40) revealed the presence of several distinct affinity-labeled receptor species for TGF-beta in both cell types and the 80K PTH/PTHrp receptors in ROS 17/2.8 cells. The affinity-labeled TGF-beta receptor species were insensitive to excess PTHrp and PTH peptides, and the 80K PTH/PTHrp receptors were insensitive to excess TGF-beta, indicating that PTHrp and TGF-beta do not cross-react with respect to receptor binding for interaction with these cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Kikuchi
H
H
Department of Radiology and Nuclear Medicine, University Hospital, Sakyo, Japan.
Shigeno
C
C
Lee
K
K
Ohta
S
S
Shiomi
K
K
Ikeda
T
T
Sone
T
T
Dokoh
S
S
Konishi
J
J
D000345:Affinity Labels; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D003114:Colony-Forming Units Assay; D003432:Cross-Linking Reagents; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology; D005353:Fibronectins / Q000096:biosynthesis; D046911:Macromolecular Substances; D044162:Parathyroid Hormone-Related Protein; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D011506:Proteins / Q000737:chemistry / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000473:pathology
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D005353:Fibronectins; D046911:Macromolecular Substances; D044162:Parathyroid Hormone-Related Protein; D010455:Peptides; D011506:Proteins; D016212:Transforming Growth Factor beta; D004815:Epidermal Growth Factor