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Dickkopf-1 negatively regulates the expression of osteoprotegerin, a key osteoclastogenesis inhibitor, by sequestering Lrp6 in primary and metastatic lytic bone lesions

View Article: PubMed Central - PubMed

ABSTRACT

Recently, an inverse role for Wnt signaling in the development of osteoclasts in the bone was demonstrated. In the present study, we examined whether there is a commonality in the mechanism of bone resorption and lysis that occur in a diverse set of bone metastatic lesions, as well as in primary bone lesions. Compared with control bone tissue and bone biopsies from patients with nonmetastatic primary tumors (i.e., breast carcinoma, lung adenocarcinoma, and prostate carcinoma), patients with bone metastatic lesions from the three aforementioned primary tumors, as well as osteolytic lesions obtained from the bone biopsies of patients with multiple myeloma, demonstrated an upregulated expression of the glycoprotein Dickkopf-1 at both the mRNA and protein levels. Additionally, by coimmunoprecipitation, Dickkopf-1 pulled-down low-density lipoprotein receptor-related protein 6 (Lrp6), which is a key downstream effector of the Wnt signaling pathway. The expression of Lrp6 was unaltered in the osteometastatic lesions. This negative regulation was associated with a lowered expression of osteoprotegerin in the osteometastatic lesions, an observation that was previously reported to promote osteoclastogenesis. These findings provide a common mechanism for the inverse relationship between the Wnt signaling pathway and the development of primary or metastatic bone lesions. Pharmacological modulation of the Wnt signaling pathway might benefit the clinical management of primary and metastatic bone lesions.

No MeSH data available.


(A and B) Coimmunoprecipitation of the pulled-down Dickkopf-1–Lrp6 immunoblots with either antibody shows the binding. (C) Unaltered levels of Lrp6 in the osteometastatic lesions compared with the controls. All of the panels are representative Western blots. (A and B) In the far left and middle panel, the immunoprecipitated lysates were blotted for Dickkopfl-1 and Lrp6, respectively. GAPDH was used to demonstrate equal loading of the loading lanes. The histograms below each blot are the quantitative intensities of the protein signals. (C) The far right panel shows the expression of Lrp6 in the control, primary, and metastatic lysates. β-actin was used as a loading control. The histogram below represents the quantified intensities of the protein signals. All of the assays were performed in triplicate from pooled samples. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
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Figure 3: (A and B) Coimmunoprecipitation of the pulled-down Dickkopf-1–Lrp6 immunoblots with either antibody shows the binding. (C) Unaltered levels of Lrp6 in the osteometastatic lesions compared with the controls. All of the panels are representative Western blots. (A and B) In the far left and middle panel, the immunoprecipitated lysates were blotted for Dickkopfl-1 and Lrp6, respectively. GAPDH was used to demonstrate equal loading of the loading lanes. The histograms below each blot are the quantitative intensities of the protein signals. (C) The far right panel shows the expression of Lrp6 in the control, primary, and metastatic lysates. β-actin was used as a loading control. The histogram below represents the quantified intensities of the protein signals. All of the assays were performed in triplicate from pooled samples. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

Mentions: Binding assays were performed to estimate the key signaling molecules for the Wnt signaling pathway, namely Lrp6. For this assay, the binding of Lrp6 with Dickkopf-1 was assayed by coimmunoprecipitation. In all of the metastatic bone lesions, an enhanced binding of Lrp6–Dickkopf-1 was observed when the proteins were immunoprecipitated with either the Lrp6 or the Dickkopf-1 antibody (Fig. 3A and B). Control experiments, in which the primary antibodies were omitted, did not result in the pull-down of the associated protein (data not shown). Minimal binding was seen in the primary tumors without metastasis (primary tumors of the breast/lung/prostate) and in the control bone biopsies. The expression of Lrp6 in the metastatic lesions was not different from the control tissues (Fig. 3C). This observation suggests that the increased sequestration of Lrp6 by the enhanced Dickkopf-1 expression probably results in the downregulation or inhibition of Wnt signaling.


Dickkopf-1 negatively regulates the expression of osteoprotegerin, a key osteoclastogenesis inhibitor, by sequestering Lrp6 in primary and metastatic lytic bone lesions
(A and B) Coimmunoprecipitation of the pulled-down Dickkopf-1–Lrp6 immunoblots with either antibody shows the binding. (C) Unaltered levels of Lrp6 in the osteometastatic lesions compared with the controls. All of the panels are representative Western blots. (A and B) In the far left and middle panel, the immunoprecipitated lysates were blotted for Dickkopfl-1 and Lrp6, respectively. GAPDH was used to demonstrate equal loading of the loading lanes. The histograms below each blot are the quantitative intensities of the protein signals. (C) The far right panel shows the expression of Lrp6 in the control, primary, and metastatic lysates. β-actin was used as a loading control. The histogram below represents the quantified intensities of the protein signals. All of the assays were performed in triplicate from pooled samples. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4998439&req=5

Figure 3: (A and B) Coimmunoprecipitation of the pulled-down Dickkopf-1–Lrp6 immunoblots with either antibody shows the binding. (C) Unaltered levels of Lrp6 in the osteometastatic lesions compared with the controls. All of the panels are representative Western blots. (A and B) In the far left and middle panel, the immunoprecipitated lysates were blotted for Dickkopfl-1 and Lrp6, respectively. GAPDH was used to demonstrate equal loading of the loading lanes. The histograms below each blot are the quantitative intensities of the protein signals. (C) The far right panel shows the expression of Lrp6 in the control, primary, and metastatic lysates. β-actin was used as a loading control. The histogram below represents the quantified intensities of the protein signals. All of the assays were performed in triplicate from pooled samples. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
Mentions: Binding assays were performed to estimate the key signaling molecules for the Wnt signaling pathway, namely Lrp6. For this assay, the binding of Lrp6 with Dickkopf-1 was assayed by coimmunoprecipitation. In all of the metastatic bone lesions, an enhanced binding of Lrp6–Dickkopf-1 was observed when the proteins were immunoprecipitated with either the Lrp6 or the Dickkopf-1 antibody (Fig. 3A and B). Control experiments, in which the primary antibodies were omitted, did not result in the pull-down of the associated protein (data not shown). Minimal binding was seen in the primary tumors without metastasis (primary tumors of the breast/lung/prostate) and in the control bone biopsies. The expression of Lrp6 in the metastatic lesions was not different from the control tissues (Fig. 3C). This observation suggests that the increased sequestration of Lrp6 by the enhanced Dickkopf-1 expression probably results in the downregulation or inhibition of Wnt signaling.

View Article: PubMed Central - PubMed

ABSTRACT

Recently, an inverse role for Wnt signaling in the development of osteoclasts in the bone was demonstrated. In the present study, we examined whether there is a commonality in the mechanism of bone resorption and lysis that occur in a diverse set of bone metastatic lesions, as well as in primary bone lesions. Compared with control bone tissue and bone biopsies from patients with nonmetastatic primary tumors (i.e., breast carcinoma, lung adenocarcinoma, and prostate carcinoma), patients with bone metastatic lesions from the three aforementioned primary tumors, as well as osteolytic lesions obtained from the bone biopsies of patients with multiple myeloma, demonstrated an upregulated expression of the glycoprotein Dickkopf-1 at both the mRNA and protein levels. Additionally, by coimmunoprecipitation, Dickkopf-1 pulled-down low-density lipoprotein receptor-related protein 6 (Lrp6), which is a key downstream effector of the Wnt signaling pathway. The expression of Lrp6 was unaltered in the osteometastatic lesions. This negative regulation was associated with a lowered expression of osteoprotegerin in the osteometastatic lesions, an observation that was previously reported to promote osteoclastogenesis. These findings provide a common mechanism for the inverse relationship between the Wnt signaling pathway and the development of primary or metastatic bone lesions. Pharmacological modulation of the Wnt signaling pathway might benefit the clinical management of primary and metastatic bone lesions.

No MeSH data available.