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Parathyroid-hormone-related protein signaling mechanisms in lung carcinoma growth inhibition.

Montgrain PR, Phun J, Vander Werff R, Quintana RA, Davani AJ, Hastings RH - Springerplus (2015)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Medicine Service, Pulmonary and Critical Care Division, Department of Medicine, VA San Diego Healthcare System, UC San Diego, San Diego, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Combined ERK1 and ERK2 siRNA knockdown vs. proliferation. a H1944 cells were transfected with equal quantities of ERK1 siRNA and ERK2 siRNA for 48 h, and placed into fresh media for the indicated times up to an additional 72 h. The control consisted of NTC siRNA at the same total oligonucleotide concentration. Combined ERK1/2 knockdown led to 20–50% reduction in pERK1 levels and 50–75% reduction in pERK2 over the course of the 72 h. b The effect of combined ERK1/2 knockdown on H1944 cell proliferation was similar to that of ERK2 knockdown (Figure 4). Overall, the effects on growth were minimal except for a 13% decrease in proliferation of PTHrP-positive H1944 cells at 72 h. *P < 0.05 vs. control siRNA by paired t test.
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Fig6: Combined ERK1 and ERK2 siRNA knockdown vs. proliferation. a H1944 cells were transfected with equal quantities of ERK1 siRNA and ERK2 siRNA for 48 h, and placed into fresh media for the indicated times up to an additional 72 h. The control consisted of NTC siRNA at the same total oligonucleotide concentration. Combined ERK1/2 knockdown led to 20–50% reduction in pERK1 levels and 50–75% reduction in pERK2 over the course of the 72 h. b The effect of combined ERK1/2 knockdown on H1944 cell proliferation was similar to that of ERK2 knockdown (Figure 4). Overall, the effects on growth were minimal except for a 13% decrease in proliferation of PTHrP-positive H1944 cells at 72 h. *P < 0.05 vs. control siRNA by paired t test.

Mentions: To avoid the confounding influence of ERK2 upregulation, we combined the siRNA treatments to knock down both isoforms. ERK1 plus ERK2 knockdown decreased both pERK1 and pERK2 levels over 72 h. The reduction in pERK1 ranged from 20 to 50% knockdown, while pERK2 decreased by 50–75%, depending on the elapsed time after the transfection. Both ERK siRNAs gradually lost their knockdown efficiency over time, but pERK levels were still reduced compared to the control siRNA at 72 h (Figure 6a).Figure 6


Parathyroid-hormone-related protein signaling mechanisms in lung carcinoma growth inhibition.

Montgrain PR, Phun J, Vander Werff R, Quintana RA, Davani AJ, Hastings RH - Springerplus (2015)

Combined ERK1 and ERK2 siRNA knockdown vs. proliferation. a H1944 cells were transfected with equal quantities of ERK1 siRNA and ERK2 siRNA for 48 h, and placed into fresh media for the indicated times up to an additional 72 h. The control consisted of NTC siRNA at the same total oligonucleotide concentration. Combined ERK1/2 knockdown led to 20–50% reduction in pERK1 levels and 50–75% reduction in pERK2 over the course of the 72 h. b The effect of combined ERK1/2 knockdown on H1944 cell proliferation was similar to that of ERK2 knockdown (Figure 4). Overall, the effects on growth were minimal except for a 13% decrease in proliferation of PTHrP-positive H1944 cells at 72 h. *P < 0.05 vs. control siRNA by paired t test.
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Related In: Results  -  Collection

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Fig6: Combined ERK1 and ERK2 siRNA knockdown vs. proliferation. a H1944 cells were transfected with equal quantities of ERK1 siRNA and ERK2 siRNA for 48 h, and placed into fresh media for the indicated times up to an additional 72 h. The control consisted of NTC siRNA at the same total oligonucleotide concentration. Combined ERK1/2 knockdown led to 20–50% reduction in pERK1 levels and 50–75% reduction in pERK2 over the course of the 72 h. b The effect of combined ERK1/2 knockdown on H1944 cell proliferation was similar to that of ERK2 knockdown (Figure 4). Overall, the effects on growth were minimal except for a 13% decrease in proliferation of PTHrP-positive H1944 cells at 72 h. *P < 0.05 vs. control siRNA by paired t test.
Mentions: To avoid the confounding influence of ERK2 upregulation, we combined the siRNA treatments to knock down both isoforms. ERK1 plus ERK2 knockdown decreased both pERK1 and pERK2 levels over 72 h. The reduction in pERK1 ranged from 20 to 50% knockdown, while pERK2 decreased by 50–75%, depending on the elapsed time after the transfection. Both ERK siRNAs gradually lost their knockdown efficiency over time, but pERK levels were still reduced compared to the control siRNA at 72 h (Figure 6a).Figure 6

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Medicine Service, Pulmonary and Critical Care Division, Department of Medicine, VA San Diego Healthcare System, UC San Diego, San Diego, USA.

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.

No MeSH data available.


Related in: MedlinePlus