Marilyn L. G. Lamm

Emerging Roles for Hedgehog-Patched-Gli Signal Transduction in Reproduction

Abstract Hedgehog (Hh) proteins are expressed during vertebrate development in some tissues with inductive properties and at epithelial-mesenchymal boundaries in several developing organs, including the lung, gut, hair follicle, and tooth. The Hh signaling pathway is highly conserved, and important clues to understanding the mechanism of Hh signal transduction in vertebrates have come from studies in Drosophila. In recent years, Hh signaling has been recognized during embryonic development and in some cases during postnatal life in several mammalian tissues whose functions are essential for reproduction, including the gonads, uterus, and hormonally responsive accessory sex glands such as the prostate and mammary gland. The role of the pathway in these tissues is highly reminiscent of its role at epithelial-mesenchymal-stromal boundaries in other organ systems, which has provided a framework within which to explore Hh signaling in tissues that function in reproduction. Some features unique to these tissues are emerging, including a role in proliferation and differentiation of male germline cells in mammals and apparent influences of sex steroids on Hh signaling. However, many questions remain about the function of Hh signaling in the gonads, uterus, prostate, and mammary gland, including factors regulating the signal transduction pathway, identification of downstream target genes, and roles for Hh signaling in diseases involving these tissues.

Luteinizing hormone/choriogonadotropin receptor-mediated activation of heterotrimeric guanine nucleotide binding proteins in ovarian follicular membranes

The LH/CG receptor signals to adenylyl cyclase via the stimulatory heterotrimeric GTP binding regulatory protein, Gs, and to phospholipase C and potentially to other effectors, such as ion channels, via a G protein or proteins that have not been identified in gonadal cells. To identify G proteins activated in a physiological membrane environment upon LH/CG receptor activation, we used the ability of activated G proteins to bind GTP and incubated ovarian follicular membranes with the photoaffinity GTP analog, P3-(4-azidoanilido)-P1-5′-GTP ([32P]AAGTP). Results showed that human CG (hCG) stimulated the binding of [32P]AAGTP to a 45-kDa protein(s) in follicular membranes that comigrated with immunoreactive Gαs, Gαq/11, and Gα13. When Gα proteins were immunoprecipitated from Triton X-100 solubilized membrane extracts after photoaffinity labeling with [32P]AAGTP, a time-dependent increase in hCG-dependent [32P]AAGTP binding to Gαs, Gαq/11, and Gαi was detected. hCG-dependent [32P]AAGTP binding to Gα13 was also...

Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiation

BackgroundSonic hedgehog (Shh) and components of its signalling pathway have been identified in human prostate carcinoma and increased levels of their expression appear to correlate with disease progression and metastasis. The mechanism through which Shh signalling could promote metastasis in bone, the most common site for prostate carcinoma metastasis, has not yet been investigated. The present study determined the effect of Shh signalling between prostate cancer cells and pre-osteoblasts on osteoblast differentiation, a requisite process for new bone formation that characterizes prostate carcinoma metastasis.ResultsLNCaP human prostate cancer cells modified to overexpress Shh (designated LNShh cells) and MC3T3 mouse pre-osteoblasts were maintained as mixed populations within the same culture chamber. In this non-conventional mixed culture system, LNShh cells upregulated the expression of Shh target genes Gli1 and Patched 1 (Ptc1) in MC3T3 cells and this was inhibited by cyclopamine, a specific chemical inhibitor of hedgehog signalling. Concomitantly, MC3T3 cells exhibited time-dependent decreased cell proliferation, upregulated alkaline phosphatase Akp2 gene expression, and increased alkaline phosphatase activity indicative of early phase osteoblast differentiation. LNShh cell-induced differentiation was inhibited in MC3T3 cells stably transfected with a dominant negative form of Gli1, a transcription factor that mediates Shh signalling. Interestingly, LNShh cells did not significantly increase the endogenous expression of the osteoblast differentiation transcription factor Runx2 and its target genes osteocalcin and osteopontin. Consistent with these results, exogenous Shh peptide did not upregulate Runx2 expression in MC3T3 cells. However, Runx2 levels were increased in MC3T3 cells by ascorbic acid, a known stimulator of osteoblast differentiation.ConclusionAltogether, these data demonstrate that Shh-expressing prostate cancer cells can directly and specifically induce differentiation in pre-osteoblasts via a Gli1-dependent mechanism that does not require transcriptional upregulation of Runx2. Paracrine activation of the Shh pathway in osteoblast progenitors and subsequent induction of osteoblast differentiation could be a mechanism through which high levels of Shh expression in prostate carcinoma contribute to bone metastasis. Targeting of paracrine Shh signalling may provide an effective therapeutic strategy against prostate carcinoma metastasis in bone.

A Proliferative Effect of Transforming Growth Factor-β1 on a Human Prostate Cancer Cell Line, TSU-Pr1

Transforming growth factor-β (TGF-β) is growth inhibitory to many malignant cells, including prostate cancer cells. The present study reports an unusual observation in that TGF-β is growth stimulatory to a human prostate cancer cell line, TSU-Pr1. The TSU-Pr1 line is highly aggressive and exhibits a rapid rate of proliferation in culture. These cells underwent further proliferation in response to TGF-β1. Both type I and II receptors to TGF-β (TβR-I, TβR-II) are expressed in TSU-Pr1 cells. Activation of a luciferase reporter gene, which contains a TGF-β response element, confirmed that the TGF-β receptors in TSU-Pr1 cells were functional. RT-PCR analysis and an ELISA assay determined that TSU-Pr1 cells secreted TGF-β. In conclusion, TSU-Pr1 cells contain functional TGF-β receptors but instead of the usual growth inhibition by TGF-β1, these cells undergo proliferation. The present observation provides a proliferative role of TGF-β in TSU-Pr1 cells, which may play a part in the aggressive phenotype of these ...

Transforming Growth Factor-β1-Induced Proliferation of the Prostate Cancer Cell Line, TSU-Pr1: The Role of Platelet-Derived Growth Factor1

The results of our previous study revealed that transforming growth factor-β1 (TGFβ1) stimulated proliferation of the prostate cancer cell line, TSU-Pr1. This observation is unexpected, for TGFβ usually inhibits proliferation in prostate cancer cells. The present study examines possible mechanisms through which TGFβ1 induces this proliferation. We postulate that TGFβ1 action is mediated through an indirect mechanism by inducing the expression of platelet-derived growth factor (PDGF), which, in turn, stimulates proliferation. The TGFβ1-induced proliferation can be abrogated by treatment with a PDGF-neutralizing antibody. Treatment with exogenous PDGF significantly increased TSU-Pr1 proliferation. Finally, treatment of TSU-Pr1 cells with TGFβ1 resulted in an increase in PDGF secretion. These results indicate that TGFβ1-induced proliferation in TSU-Pr1 cells is at least mediated through an increased secretion of PDGF.

Transforming Growth Factor-β1 Inhibits Membrane Association of Protein Kinase Cα in a Human Prostate Cancer Cell Line, PC31

The postreceptor signaling pathway(s) that mediates the effects of transforming growth factor-beta1 (TGF-beta1) is incompletely understood. The present study investigated the involvement of protein kinase C (PKC) in the growth-inhibitory action of TGF-beta1 in PC3, a human prostate cancer cell line. PKC alpha, the only conventional PKC isoform detected in PC3 cells, appeared to be constitutively active based on its presence in both Triton-soluble membrane fraction and cytosol. However, levels of membrane-associated PKC alpha were decreased by a growth-inhibitory dose of TGF-beta1. The response to TGF-beta1 was rapid (within 5 min), time dependent, isoform specific, and occurred without apparent changes in levels of total PKC alpha protein. TGF-beta1 also decreased the levels of membrane-associated PKC activity coincident with its inhibitory effect on PKC alphas membrane association. Inhibition of PKC activity appeared to be associated with growth inhibition in PC3 cells, because chelerythrine (a specific PKC inhibitor) likewise decreased cell proliferation. Taken together, our data suggest that inhibition of PKC activity, at least in part due to inactivation of PKC alpha, is an early event associated with TGF-beta1 postreceptor signaling that might mediate suppression of cell proliferation.

Noncanonical regulation of the Hedgehog mediator GLI1 by c-MYC in Burkitt lymphoma.

Although Hedgehog signaling plays a major role in GLI1 transcription, there is now evidence suggesting that other pathways/genes, such as c-MYC, may also regulate GLI1 expression. We initiated studies in Burkitt lymphoma cells, which constitutively express c-MYC due to a chromosomal translocation, to determine whether Hedgehog or c-MYC regulates GLI1 expression. We show that all Burkitt lymphoma cell lines tested express GLI1, PTCH1, and SMO and that five of six Burkitt lymphomas express GLI1. Exposure to Sonic or Indian Hedgehog or cyclopamine (SMO inhibitor) does not modulate GLI1 expression, cell proliferation, or apoptosis in most Burkitt lymphoma cell lines. Sequence analysis of PTCH1, SMO, and SuFu failed to show mutations that might explain the lack of Hedgehog responsiveness, and we did not detect primary cilia, which may contribute to it. We show that c-MYC interacts with the 5′-regulatory region of GLI1, using chromatin immunoprecipitation (ChIP) assay, and E-box–dependent transcriptional activation of GLI1 by c-MYC in NIH3T3 and HeLa cells. The c-MYC small-molecule inhibitor 10058-F4 downregulates GLI1 mRNA and protein and reduces the viability of Burkitt lymphoma cells. Inhibition of GLI1 by GANT61 increases apoptosis and reduces viability of some Burkitt lymphoma cells. Collectively, our data provide evidence that c-MYC directly regulates GLI1 and support an antiapoptotic role for GLI1 in Burkitt lymphoma. Burkitt lymphoma cells do not seem to be Hedgehog responsive. These findings suggest a mechanism for resistance to SMO inhibitors and have implications for using SMO inhibitors to treat human cancers. Mol Cancer Res; 11(6); 604–15. ©2013 AACR.

Hormonal regulation of PKC-δ protein and mRNA levels in the rabbit corpus luteum ☆

We have previously reported that rabbit corpora lutea exhibit a prominent phosphorylated substrate protein at 76 kDa which corresponds to the autophosphorylated form of protein kinase C (PKC) delta and that the expression of PKC-delta protein is increased in rabbit corpora lutea of pseudopregnancy at least 2-fold when serum estrogen levels are raised by the presence of an estrogen implant inserted at the time of human chorionic gonadotropin (hCG)-induced ovulation. The purpose of the experiments described herein was to evaluate further the hormonal regulation of PKC-delta in the rabbit corpus luteum. Results demonstrate that luteal PKC-delta protein and mRNA are concomitantly induced some 5-fold within 48 h in response to an ovulatory surge of hCG; that, as in corpora lutea of pseudopregnancy, luteal PKC-delta expression is relatively constant during the life span of the corpus luteum following a fertile mating; that exogenous estrogen does not modulate the induction of luteal PKC-delta during luteinization but promotes an additional two-fold increase in steady state PKC-delta mRNA (and protein) levels in corpora lutea by day 10 of pseudopregnancy; and that luteal PKC-delta expression can be abruptly and reversibly modulated upon withdrawal and subsequent replacement of an estrogen implant to pseudopregnant rabbits. These results demonstrate that an ovulatory surge of luteinizing hormone induces the expression of PKC-delta mRNA and protein in rabbit corpora lutea, and that once the corpus luteum becomes estrogen responsive, estrogen then regulates expression of PKC-delta mRNA and protein.

Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation.

BackgroundInduction of osteoblast differentiation by paracrine Sonic hedgehog (Shh) signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts.ResultsIn the presence of exogenous ascorbic acid (AA), a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells) formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis inhibitor 3,4-dehydro-L-proline.ConclusionsMatrix collagen, formed by osteoblasts in the presence of AA, potentiates Shh signaling between Shh-expressing prostate cancer cells and osteoblasts. Collagen and Shh signaling exert a synergistic effect on osteoblast differentiation, a defining event in prostate carcinoma bone metastasis. Investigations into paracrine interactions among prostate cancer cells, osteoblasts, and osteoblast-synthesized matrix proteins advance our understanding of mechanisms contributing to prostate cancer bone metastasis.

Epidermal Growth Factor-Induced Heterologous Desensitization of the Luteinizing Hormone/Choriogonadotopin Receptor in a Cell-Free Membrane Preparation Is Associated with the Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor1

Epidermal growth factor (EGF) attenuated hCG-stimulated adenylyl cyclase activity in rat luteal and follicular membranes. H7, an equipotent serine/threonine protein kinase inhibitor of cAMP-dependent protein kinases, cGMP-dependent protein kinases, and lipid-dependent protein kinase C, did not effect the ability of EGF to decrease hCG-responsive adenylyl cyclase activity, suggesting that a serine/threonine phosphorylation event catalyzed by these kinases was not critically involved in EGF-induced desensitization. Likewise, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa luteal membrane protein, which exhibited immunoreactivity with an antibody against Gi alpha, did not hinder the ability of EGF to attenuate hCG-stimulated adenylyl cyclase activity, indicating that Gi did not mediate EGF-induced desensitization. Rather, EGF-induced heterologous desensitization of LH/CG receptor in ovarian membranes was closely associated with the specific and prominent tyrosine phosphorylation of the 170-kDa EGF receptor. Both EGF-stimulated autophosphorylation of EGF receptor and EGF-induced LH/CG receptor desensitization were attenuated by genistein, a tyrosine kinase inhibitor. These results suggest that tyrosine phosphorylation of the 170-kDa EGF receptor is a necessary component of the signaling pathway in EGF-induced heterologous desensitization of the LH/CG receptor.