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Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiation.

Zunich SM, Douglas T, Valdovinos M, Chang T, Bushman W, Walterhouse D, Iannaccone P, Lamm ML - Mol. Cancer (2009)

Bottom Line: 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.Altogether, 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Northwestern University Feinberg School of Medicine, Children's Memorial Research Center, Chicago, IL 60614, USA. SZunich@childrensmemorial.org

ABSTRACT

Background: Sonic 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.

Results: LNCaP 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.

Conclusion: Altogether, 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.

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Dominant negative Gli1 inhibits paracrine Shh signalling and osteoblast differentiation. (A) MC3T3 cells were stably transfected with human GLI1 cDNA with deleted region containing the trans activation domain: pCMV-GL1(-)TAD. Expression of mouse endogenous Gli1 (mGli1) and human GLI1-TAD transgene in parental MC3T3 cells and transfected cells, designated M-TAD cells, were determined by conventional RT-PCR analysis. The mouse gene for ribosomal protein 19 (mrpl19) was used as housekeeping gene. (B) Hematoxylin staining of single cultures of MC3T3 and M-TAD cells are shown in panels a and b, respectively. Fluorescent microscopy images of 14-day mixed cultures of MC3T3 and LNShh cells or M-TAD and LNShh cells are shown in panels c and d, respectively. The photomicrograph of mixed culture of MC3T3 and LNShh cells shown in panel c is the same as that shown in Figure 1 panel c, except only phalloidin staining and GFP expression are revealed here. Scale bars: 25 μm (a, b) and 50 μm (c, d). (C) MC3T3 cells or M-TAD cells were grown for 14 days in 6-well plates with culture inserts containing either LNCaP or LNShh cells (separate co-cultures). Expression of Gli1 and Ptc1 in MC3T3 or M-TAD cells co-cultured with LNShh cells (filled bars) relative to those co-cultured with control LNCaP cells (open bars) were compared. Results are representative of 2 independent experiments. (D) Mixed cultures of MC3T3 and M-TAD cells with either LNCaP or LNShh cells were grown in chamber slides for 14 days and stained for ALP activity (panels a, b and c) followed by hematoxylin staining (panels d, e and f). Scale bars: 50 μm.
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Figure 4: Dominant negative Gli1 inhibits paracrine Shh signalling and osteoblast differentiation. (A) MC3T3 cells were stably transfected with human GLI1 cDNA with deleted region containing the trans activation domain: pCMV-GL1(-)TAD. Expression of mouse endogenous Gli1 (mGli1) and human GLI1-TAD transgene in parental MC3T3 cells and transfected cells, designated M-TAD cells, were determined by conventional RT-PCR analysis. The mouse gene for ribosomal protein 19 (mrpl19) was used as housekeeping gene. (B) Hematoxylin staining of single cultures of MC3T3 and M-TAD cells are shown in panels a and b, respectively. Fluorescent microscopy images of 14-day mixed cultures of MC3T3 and LNShh cells or M-TAD and LNShh cells are shown in panels c and d, respectively. The photomicrograph of mixed culture of MC3T3 and LNShh cells shown in panel c is the same as that shown in Figure 1 panel c, except only phalloidin staining and GFP expression are revealed here. Scale bars: 25 μm (a, b) and 50 μm (c, d). (C) MC3T3 cells or M-TAD cells were grown for 14 days in 6-well plates with culture inserts containing either LNCaP or LNShh cells (separate co-cultures). Expression of Gli1 and Ptc1 in MC3T3 or M-TAD cells co-cultured with LNShh cells (filled bars) relative to those co-cultured with control LNCaP cells (open bars) were compared. Results are representative of 2 independent experiments. (D) Mixed cultures of MC3T3 and M-TAD cells with either LNCaP or LNShh cells were grown in chamber slides for 14 days and stained for ALP activity (panels a, b and c) followed by hematoxylin staining (panels d, e and f). Scale bars: 50 μm.

Mentions: Both parental MC3T3 and the M-TAD cells expressed endogenous mouse Gli1 mRNA (Figure 4A, lanes 1 and 2). However, only M-TAD cells expressed the message for the dominant negative human GLI(-)TAD transgene (Figure 4A, lanes 3 and 4). Surprisingly, the phenotype of M-TAD cells appeared different from that of parental MC3T3 cells. When grown to confluence, MC3T3 cells changed from a spindle-shaped phenotype to a cuboidal morphology with generally round nuclei indicative of the differentiated state (Figure 4B, panel a). M-TAD cells, on the other hand, retained their fusiform shape with nuclei elongated along the long cell axis consistent with an immature osteoblast state (Figure 4B, panel b). As with MC3T3 cells, M-TAD cells in mixed culture with LNShh cells formed a stroma surrounding human prostate cancer cells although their stroma appeared to be more compact (Figure 4B, panels c and d).


Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiation.

Zunich SM, Douglas T, Valdovinos M, Chang T, Bushman W, Walterhouse D, Iannaccone P, Lamm ML - Mol. Cancer (2009)

Dominant negative Gli1 inhibits paracrine Shh signalling and osteoblast differentiation. (A) MC3T3 cells were stably transfected with human GLI1 cDNA with deleted region containing the trans activation domain: pCMV-GL1(-)TAD. Expression of mouse endogenous Gli1 (mGli1) and human GLI1-TAD transgene in parental MC3T3 cells and transfected cells, designated M-TAD cells, were determined by conventional RT-PCR analysis. The mouse gene for ribosomal protein 19 (mrpl19) was used as housekeeping gene. (B) Hematoxylin staining of single cultures of MC3T3 and M-TAD cells are shown in panels a and b, respectively. Fluorescent microscopy images of 14-day mixed cultures of MC3T3 and LNShh cells or M-TAD and LNShh cells are shown in panels c and d, respectively. The photomicrograph of mixed culture of MC3T3 and LNShh cells shown in panel c is the same as that shown in Figure 1 panel c, except only phalloidin staining and GFP expression are revealed here. Scale bars: 25 μm (a, b) and 50 μm (c, d). (C) MC3T3 cells or M-TAD cells were grown for 14 days in 6-well plates with culture inserts containing either LNCaP or LNShh cells (separate co-cultures). Expression of Gli1 and Ptc1 in MC3T3 or M-TAD cells co-cultured with LNShh cells (filled bars) relative to those co-cultured with control LNCaP cells (open bars) were compared. Results are representative of 2 independent experiments. (D) Mixed cultures of MC3T3 and M-TAD cells with either LNCaP or LNShh cells were grown in chamber slides for 14 days and stained for ALP activity (panels a, b and c) followed by hematoxylin staining (panels d, e and f). Scale bars: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Dominant negative Gli1 inhibits paracrine Shh signalling and osteoblast differentiation. (A) MC3T3 cells were stably transfected with human GLI1 cDNA with deleted region containing the trans activation domain: pCMV-GL1(-)TAD. Expression of mouse endogenous Gli1 (mGli1) and human GLI1-TAD transgene in parental MC3T3 cells and transfected cells, designated M-TAD cells, were determined by conventional RT-PCR analysis. The mouse gene for ribosomal protein 19 (mrpl19) was used as housekeeping gene. (B) Hematoxylin staining of single cultures of MC3T3 and M-TAD cells are shown in panels a and b, respectively. Fluorescent microscopy images of 14-day mixed cultures of MC3T3 and LNShh cells or M-TAD and LNShh cells are shown in panels c and d, respectively. The photomicrograph of mixed culture of MC3T3 and LNShh cells shown in panel c is the same as that shown in Figure 1 panel c, except only phalloidin staining and GFP expression are revealed here. Scale bars: 25 μm (a, b) and 50 μm (c, d). (C) MC3T3 cells or M-TAD cells were grown for 14 days in 6-well plates with culture inserts containing either LNCaP or LNShh cells (separate co-cultures). Expression of Gli1 and Ptc1 in MC3T3 or M-TAD cells co-cultured with LNShh cells (filled bars) relative to those co-cultured with control LNCaP cells (open bars) were compared. Results are representative of 2 independent experiments. (D) Mixed cultures of MC3T3 and M-TAD cells with either LNCaP or LNShh cells were grown in chamber slides for 14 days and stained for ALP activity (panels a, b and c) followed by hematoxylin staining (panels d, e and f). Scale bars: 50 μm.
Mentions: Both parental MC3T3 and the M-TAD cells expressed endogenous mouse Gli1 mRNA (Figure 4A, lanes 1 and 2). However, only M-TAD cells expressed the message for the dominant negative human GLI(-)TAD transgene (Figure 4A, lanes 3 and 4). Surprisingly, the phenotype of M-TAD cells appeared different from that of parental MC3T3 cells. When grown to confluence, MC3T3 cells changed from a spindle-shaped phenotype to a cuboidal morphology with generally round nuclei indicative of the differentiated state (Figure 4B, panel a). M-TAD cells, on the other hand, retained their fusiform shape with nuclei elongated along the long cell axis consistent with an immature osteoblast state (Figure 4B, panel b). As with MC3T3 cells, M-TAD cells in mixed culture with LNShh cells formed a stroma surrounding human prostate cancer cells although their stroma appeared to be more compact (Figure 4B, panels c and d).

Bottom Line: 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.Altogether, 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Northwestern University Feinberg School of Medicine, Children's Memorial Research Center, Chicago, IL 60614, USA. SZunich@childrensmemorial.org

ABSTRACT

Background: Sonic 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.

Results: LNCaP 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.

Conclusion: Altogether, 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.

Show MeSH
Related in: MedlinePlus