Abstract
Parathyroid hormone-related protein (PTHrP) inhibits proliferation of several lung cancer cell lines, but the signaling mechanism has not been established. This study tested the hypotheses that growth inhibition is mediated through the PTHrP receptor, PTH1R, and that the process is modified by ERK activation. PTHrP-positive and negative clones of H1944 lung adenocarcinoma cells underwent stable PTH1R knockdown with lentiviral shRNA or transient transfection with ERK1 and ERK2 siRNA. Alternatively, cells were treated with 8-CPT cAMP, 8-CPT 2′-O-methyl cAMP, and N-6-phenyl cAMP analogs. H1944 cells expressing ectopic PTHrP showed 20–40% decrease in proliferation compared to the PTHrP-negative cells in the presence of normal levels of PTH1R (P < 0.01). PTH1R knockdown eliminated this difference and increased cell proliferation regardless of PTHrP status. The three cAMP analogs each inhibited proliferation over 5 days by 30–40%. ERK2 knockdown inhibited proliferation of PTHrP-positive cells alone and in combination with ERK1 knockdown. The growth inhibition mediated by cAMP analogs was unaffected by ERK1 knockdown. In conclusion, ectopic expression of PTHrP 1–87 inhibits H1944 cell proliferation. PTH1R knockdown blocks this effect and stimulates proliferation, indicating that the ligand exerts anti-mitogenic effects. cAMP, the second messenger for PTH1R also inhibits proliferation and activates ERK. PTHrP growth inhibition may be opposed by concomitant ERK activation.
Highlights
The amino-terminal region of parathyroid hormonerelated protein (PTHrP), residues 1–34, shares structural and functional features with the same region of parathyroid hormone (PTH)
Role of PTH1R PTH1R knockdown Studies utilized three H1944 cell clones that make 600– 700 pg PTHrP 1–34/μg cell protein/24 h as a result of stable transfection with the PTHrP 1–87 expression plasmid (Hastings et al 2009). Cells from these clones were transduced with PTH1R shRNA particles or non-target control (NTC) particles to generate three PTHrP-positive, PTH1R knockdown clones and three clones with intact PTH1R
By MTS assay, the PTH1R knockdown cells nearly tripled in number over a 2 days period, a 37 ± 1% faster growth rate when compared to the NTC-transfected cells, which only doubled in number over the same period (Figure 1b, P < 0.01)
Summary
The amino-terminal region of parathyroid hormonerelated protein (PTHrP), residues 1–34, shares structural and functional features with the same region of parathyroid hormone (PTH). PTHrP 1–34 and PTH 1–34 activate the same G protein-coupled receptor, PTH1R. Mid-molecule PTHrP peptides (residues 38–94) and carboxyl-terminal PTHrP peptides (residues 107–141) have biologic activities that are unique from the effects of PTHrP 1–34 (Orloff et al 1994). Mid-molecule PTHrP fragments can regulate growth in breast cancer cells (Luparello et al 2001), while the carboxyl domain affects function and proliferation in osteoclasts (Zheng et al 1994). The non-amino effects of PTHrP are presumed to result from binding to other cell-surface receptors, but such receptors have yet to be discovered (Orloff et al 1994). A variety of Montgrain et al SpringerPlus (2015) 4:268 possible mechanisms must be considered to understand the effects of PTHrP in a given tissue or cell type
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