Protein kinase C{gamma} mediates regulation of proliferation by the serotonin 5-hydroxytryptamine receptor 2B

Abstract

Activation of the 5-hydroxytryptamine receptor 2B (5-HT(2B)), a G(q/11) protein-coupled receptor, results in proliferation of various cell types. The 5-HT(2B) receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT(2B) receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT(2B) receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by LY294002 decreased base-line proliferation but had no effect on 5-HT(2B) receptor-mediated proliferation. Proliferation of ICC through the 5-HT(2B) receptor was inhibited by the phospholipase C inhibitor U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the alpha, beta, gamma, and micro protein kinase C (PKC) inhibitor Gö6976, and the alpha, beta, gamma, delta, and zeta PKC inhibitor Gö6983 inhibited 5-HT(2B) receptor-mediated proliferation, indicating the involvement of PKC alpha, beta, or gamma. Of all the PKC isoforms blocked by Gö6976, PKCgamma and micro mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT(2B) receptor activation in primary cell cultures obtained from PKCgamma(-/-) mice did not result in a proliferative response, further indicating the requirement for PKCgamma in the proliferative response to 5-HT(2B) receptor activation. The data demonstrate that the 5-HT(2B) receptor-induced proliferative response of ICC is through phospholipase C, [Ca(2+)](i), and PKCgamma, implicating this PKC isoform in the regulation of cellular proliferation.

FIGURE 1.

The 5-HT_2B receptor agonist BW723C86 increases proliferation. The 5-HT_2B agonist BW723C86 (50 nm) increased the number of proliferating ICC. Control, 18.6 ± 0.7%; 50 nm BW, 23.9 ± 1.3%. The data are expressed as the means ± S.E. p < 0.05 by paired t test (n = 30).

FIGURE 2.

Proliferation of ICC through the 5-HT_2B receptor did not require PI3′-K but was dependent upon PLC. A, addition of the PI3′-K inhibitor LY294002 (1 and 10 μm) decreased base-line proliferation of ICC. However, in the presence of LY294002 (1 and 10 μm), activation of the 5-HT_2B receptor by BW723C86 still increased proliferation of ICC. Control, 17.2 ± 2.1%; 50 nm BW, 20.5 ± 2.7%; 1 μm LY294002, 11.0 ± 2.0%; 1 μm LY294002 + 50 nm BW, 16.8 ± 2.8%; 10 μm LY294002, 3.6 ± 1.5%; 10 μm LY294002 + 50 nm BW, 11.6 ± 3.4%. The data are expressed as the means ± S.E. p < 0.05 by Tukey-Kramer multiple comparisons test (n = 6). B, U73122 (1 and 10 μm) did not affect base-line proliferation. However, in the presence of 1 and 10 μm U73122, there was no proliferative response to BW723C86. Control, 19.8 ± 1.4%; 50 nm BW, 29.6 ± 4.4%; 1 μm U73122, 24.3 ± 5.0%; 1 μm U73122 + 50 nm BW, 21.7 ± 2.4%; 10 μm U73122, 16.6 ± 1.3%; 10 μm U73122 + 50 nm BW, 18.6 ± 2.0%. The data are expressed as the means ± S.E. p < 0.05 by Tukey-Kramer multiple comparisons test (n = 6).

FIGURE 3.

The inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C and the nonspecific PKC inhibitor calphostin C inhibited the proliferative effect of BW723C86 on ICC. A, 333 nm and 1 μm Xestospongin (Xesto) C inhibited the increase in proliferation of ICC induced by BW723C86. Control, 22.0 ± 1.0%; 50 nm BW, 27.2 ± 1.6; 333 nm Xestospongin C, 24.6 ± 0.8; 333 nm Xestospongin C + 50 nm BW, 27.1 ± 1.7%; 1 μm Xestospongin C, 22.9 ± 1.8%; 1 μm Xestospongin C + 50 nm BW, 19.9 ± 0.8%. The data are expressed as the means ± S.E. p < 0.05 by Tukey-Kramer multiple comparisons test (n = 6). B, 50 nm calphostin (Calph) partly inhibited the proliferative response by BW723C86, whereas 150 nm completely prevented the proliferative effect. Control, 21.6 ± 2.5; 50 nm BW, 25.2 ± 2.9; 50 nm calphostin C, 21.6 ± 2.2; 50 nm calphostin C + 50 nm BW, 24.1 ± 3.7; 150 nm calphostin C, 21.0 ± 1.9; 150 nm calphostin C + 50 nm BW, 20.7 ± 2.4. The data are expressed as the means ± S.E. p < 0.05 by Tukey-Kramer multiple comparisons test (n = 6).

FIGURE 4.

The isoform specific protein kinase C antagonists Gö6976 and Gö6983 inhibited the increase in proliferation of ICC induced by BW723C86. A, the PKCα, β, γ, and μ inhibitor Gö6976 decreased base-line proliferation of ICC. Increased proliferation by adding BW723C86 was completely inhibited in the presence of 100 nm Gö6976. Control, 16.4 ± 0.82; 50 nm BW, 21.1 ± 0.56; 100 nm Gö6976, 13.6 ± 0.68; 100 nm Gö6976 + 50 nm BW, 15 ± 0.84; n = 8, p < 0.05. B, the PKCα, β, γ, and δ inhibitor Gö6983 had no effect on base-line proliferation of ICC. 30 and 100 nm Gö6983 fully inhibited the proliferative response by BW723C86. Control, 16.9 ± 0.50; 50 nm BW, 21.9 ± 0.76; 30 nm Gö6983 15.5 ± 0.96, 30 nm Gö6983 + 50 nm BW, 15.8 ± 0.91; 100 nm Gö6983, 15.3 ± 0.78; 100 nm Gö6983 + 50 nm BW, 16.9 ± 1.26. The data are expressed as the means ± S.E. p > 0.05 by Tukey-Kramer multiple comparisons test (n = 6).

FIGURE 5.

ERK inhibitor inhibited the proliferative effect of BW723C86 on ICC. 25 μm of the drug inhibited the increase in proliferation of ICC induced by BW723C86. Control, 16 ± 1.1% proliferating ICC; 50 nm BW, 24.4 ± 1.2%, n = 4, p < 0.05; ERK inhibitor (25 μm), 16.6 ± 0.4%; ERK inhibitor (25 μm) + 50 nm BW, 16.0 ± 1.2% (n = 4). p > 0.05 by Tukey-Kramer multiple comparisons test. The data are expressed as the means ± S.E.

FIGURE 6.

The calcium-dependent PKC isoforms, PKCα, β, and PKCμ mRNAs were expressed in adult mouse jejunum muscle strips. mRNAs of the expected length were amplified from brain and jejunal muscle strips for PKCα (A, 414 nt), β (B, 557 nt), γ (C, 528 nt), and μ (D, 426 nt). Ladders, DNA molecular weight marker XIV (Roche Applied Science).

FIGURE 7.

Single ICC expressed PKCγ and μ mRNAs. A, mRNA of the expected length was amplified from nonlabeled cells (NLC) for PKCα (223 nt). The faint band slightly above the expected size seen in the ICC lane was sequenced and was found to be nonspecific amplification. B and C, PKCγ (B, 334 and 528 nt) and μ (C, 248 nt) mRNAs were amplified from ICC but not from nonlabeled cells. D, β-actin mRNA was amplified from ICC and nonlabeled cells (453 nt). E, as a control, c-Kit mRNA was amplified from ICC but not from nonlabeled cells (285 nt). The identity of all amplification products was confirmed by sequencing. Ladders, DNA molecular weight marker XIV (Roche Applied Science).

FIGURE 8.

The proliferative response of ICC by activation of the 5-HT_2B receptor is absent in PKCγ knock-out cultures. The 5-HT_2B receptor agonist BW723C86 (50 nm) did not increase proliferation in PKCγ^−/− ICC. Control, 17.9 ± 1.1%; 50 nm BW, 15.0 ± 1.1%. The data are expressed as the means ± S.E. p > 0.05 by paired t test (n = 4).