The role of protein kinase C (PKC)-alpha in endothelin-1 (ET-1)induced proliferation of human myometrial cells was investigated. Inhibition of conventional PKC with Go 6976 eliminated the proliferative effect of ET-1. Treatment of myometrial cells with an antisense oligonucleotide against PKC alpha efficiently reduced PKC alpha protein expression without effect on other PKC isoforms and resulted in the loss of ET-1-induced cell growth. Immunocytochemistry using an antibody against PKC alpha revealed that there was no PKC alpha immunoreactivity in the nuclei of quiescent nonconfluent untreated cells, whereas it is evenly distributed throughout the cytoplasm. Exposure of myometrial cells to ET-1 for 15 min caused the PKC alpha to shift towards the perinuclear area, and incubation for 60 min caused a shift towards the nucleus. These results reveal that PKC alpha is required for ET-1-induced human myometrial cell growth and suggest that targeting of PKC alpha by antisense nucleotides might be an important approach for the development of anticancer treatments.

The protein kinase C (PKC) isoenzyme expression pattern in human uterine leiomyoma was compared with that obtained in homologous myometrium distal from the tumor. The six PKC isoforms (PKCalpha, PKCbeta1, PKCbeta2, PKCdelta, PKCepsilon and PKCzeta) evidenced in the myometrium were found to be similarly expressed in leiomyoma. Quantitative immunoblotting revealed that all PKC isoforms were preferentially localized in the particulate fraction. To gain insight into the possible functional consequences of PKC expression patterns, subcellular redistribution in response to the mitogenic peptide endothelin-1 (ET-1) was studied. After stimulation with ET-1, differential redistribution occurred in leiomyoma and myometrium, suggesting a selective role of PKC isoforms in the myometrial growth process.

OBJECTIVE Factors responsible for the abnormal proliferation of myometrial cells that accompanies leiomyoma formation are unknown, although steroid hormones and peptide growth factors have been implicated. We hypothesized that endothelin-1 (ET-1) is a physiological regulator of tumor growth. DESIGN In this study, we investigated the role of ET-1 on growth of human leiomyoma cells and its synergistic effect with growth factors, as well as the signaling pathway involved in this interaction. METHODS Leiomyoma cell proliferation was assayed by [H]thymidine incorporation and cell number. Protein kinase C (PKC) isoforms were analyzed by Western blot using specific antibodies. RESULTS ET-1 on its own was unable to stimulate DNA synthesis but potentiated the leiomyoma cell growth effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), IGF-I and IGF-II. The failure of a protein tyrosine kinase (PTK) inhibitor, tyrphostin 51, to affect the potentiating effect of ET-1, supports the hypothesis of non-involvement of PTK in this process. The inhibition of PKC by calphostin C or its down-regulation by phorbol 12,13-dibutyrate (PDB) eliminated the potentiating effect of ET-1, but did not block cell proliferation induced by the growth factors alone. Five PKC isoforms (alpha, beta1, epsilon, delta and zeta) were detected in leiomyoma cells, but only phorbol ester-sensitive PKC isoforms (PKCalpha, epsilon and delta) contribute to the potentiating effect of leiomyoma cell growth by ET-1. CONCLUSIONS We have demonstrated that ET-1 potentiates leiomyoma cell proliferation to growth factors through a PKC-dependent pathway. These findings suggest a possible involvement of ET-1 in the pathogenesis of leiomyomas.

The role of protein kinase C (PKC) in contraction of the human myometrium induced by endothelin-1 (ET-1) was investigated at the end of pregnancy. The expression and subcellular distribution of PKC isoforms were examined by Western blot analysis using isoform-specific antibodies. At least three conventional PKC isoforms (cPKC; alpha, beta1, and beta2), two novel PKC isoforms (epsilon and delta), and an atypical PKC isoform (zeta) were detected in pregnant myometrium. Quantitative immunoblotting revealed that all these isoforms were mainly distributed in the particulate fraction. The lack of a calcium chelator to modify the particulate sequestration of cPKC suggests an interaction with an anchoring protein such as receptor-activated C kinase-1, which is evidenced in the particulate fraction of the pregnant myometrium. Of the six isoforms, only PKCbeta1, PKCbeta2, PKCdelta, and PKCzeta were translocated to the particulate fraction, and PKCepsilon to the cytoskeletal fraction, after stimulation with ET-1. Involvement of PKC in the ET-1-induced contractile response is supported by the inhibition caused by the PKC inhibitor calphostin C. However, we demonstrated that the selective cPKC isoform inhibitor, Gö 6976, as well as the substantial depletion of PKCbeta1 and PKCepsilon and the partial depletion of PKCalpha and PKCdelta by a long-term treatment with phorbol 12,13-dibutyrate did not prevent ET-1-induced contraction. Accordingly, our results suggest that PKCdelta and PKCzeta activation mediated ET-1-induced contraction, whereas cPKC isoforms were not implicated in the human pregnant myometrium.

The role of protein kinase C (PKC) in endothelin-1 (ET-1)-induced proliferation of human myometrial cells was investigated. ET-1 dose dependently stimulated DNA synthesis and the number of cultured myometrial cells. Inhibition of PKC by calphostin C or Ro-31-8220 or downregulation of PKC eliminated the proliferative effects of ET-1. The failure of two protein tyrosine kinase (PTK) inhibitors (tyrphostin 51 and tyrphostin 23) to affect ET-1-induced proliferation supports the hypothesis of noninvolvement of the tyrosine kinase signaling pathway in this process. The expression and distribution of PKC isoforms were examined by Western blot analysis. The five PKC isoforms (PKC-alpha, -beta1, -beta2, -zeta, -epsilon) evidenced in human myometrial tissue were found to be differentially expressed in myometrial cells, with a predominant expression of PKC-alpha and PKC-zeta. Treatment with phorbol 12, 13-dibutyrate (PDBu) resulted in the translocation of all five isoforms to the particulate fraction, whereas ET-1 induced a selective increase in particulate PKC-beta1, PKC-beta2, and PKC-epsilon. Our findings that multiple PKC isoforms are differentially responsive to ET-1 or PDBu suggest that they play distinct roles in the myometrial growth process.