DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaPM cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis

Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/-) mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr-/- murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD

Molecular implications of evolutionary differences in CHD double chromodomains

Double chromodomains occur in CHD proteins, which are ATP-dependent chromatin remodeling factors implicated in RNA polymerase II transcription regulation. Biochemical studies suggest important differences in the histone H3 tail binding of different CHD chromodomains. In human and Drosophila, CHD1 double chromodomains bind lysine 4-methylated histone H3 tail, which is a hallmark of transcriptionally active chromatin in all eukaryotes. Here, we present the crystal structure of the yeast CHD1 double chromodomains, and pinpoint their differences with that of the human CHD1 double chromodomains. The most conserved residues in these double chromodomains are the two chromoboxes that orient adjacently. Only a subset of CHD chromoboxes can form an aromatic cage for methyllysine binding, and methyllysine binding requires correctly oriented inserts. These factors preclude yeast CHD1 double chromodomains from interacting with the histone H3 tail. Despite great sequence similarity between the human CHD1 and CHD2 chromodomains, variation within an insert likely prevents CHD2 double chromodomains from binding lysine 4-methylated histone H3 tail as efficiently as in CHD1. By using the available structural and biochemical data we highlight the evolutionary specialization of CHD double chromodomains, and provide insights about their targeting capacities.status: publishe

Osteoblasts Generate Testosterone From DHEA and Activate Androgen Signaling in Prostate Cancer Cells

Bone metastasis is a complication of prostate cancer in up to 90% of men afflicted with advanced disease. Therapies that reduce androgen exposure remain at the forefront of treatment. However, most prostate cancers transition to a state whereby reducing testicular androgen action becomes ineffective. A common mechanism of this transition is intratumoral production of testosterone (T) using the adrenal androgen precursor dehydroepiandrosterone (DHEA) through enzymatic conversion by 3β- and 17β- hydroxysteroid dehydrogenases (3βHSD and 17βHSD). Given the ability of prostate cancer to form blastic metastases in bone, we hypothesized that osteoblasts might be a source of androgen synthesis. RNA expression analyses of murine osteoblasts and human bone confirmed that at least one 3βHSD and 17βHSD enzyme isoform was expressed, suggesting that osteoblasts are capable of generating androgens from adrenal DHEA. Murine osteoblasts were treated with 100- nM and 1- μM DHEA or vehicle control. Conditioned media from these osteoblasts were assayed for intermediate and active androgens by liquid chromatography- tandem mass spectrometry. As DHEA was consumed, the androgen intermediates androstenediol and androstenedione were generated and subsequently converted to T. Conditioned media of DHEA- treated osteoblasts increased androgen receptor (AR) signaling, prostate- specific antigen (PSA) production, and cell numbers of the androgen- sensitive prostate cancer cell lines C4- 2B and LNCaP. DHEA did not induce AR signaling in osteoblasts despite AR expression in this cell type. We describe an unreported function of osteoblasts as a source of T that is especially relevant during androgen- responsive metastatic prostate cancer invasion into bone. © 2021 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/169292/1/jbmr4313_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/169292/2/jbmr4313.pd

Osteoblasts Generate Testosterone From DHEA

Bone metastasis is a complication of prostate cancer in up to 90% of men afflicted with advanced disease. Therapies that reduce androgen exposure remain at the forefront of treatment. However, most prostate cancers transition to a state whereby reducing testicular androgen action becomes ineffective. A common mechanism of this transition is intratumoral production of testosterone (T) using the adrenal androgen precursor dehydroepiandrosterone (DHEA) through enzymatic conversion by 3β- and 17β- hydroxysteroid dehydrogenases (3βHSD and 17βHSD). Given the ability of prostate cancer to form blastic metastases in bone, we hypothesized that osteoblasts might be a source of androgen synthesis. RNA expression analyses of murine osteoblasts and human bone confirmed that at least one 3βHSD and 17βHSD enzyme isoform was expressed, suggesting that osteoblasts are capable of generating androgens from adrenal DHEA. Murine osteoblasts were treated with 100- nM and 1- μM DHEA or vehicle control. Conditioned media from these osteoblasts were assayed for intermediate and active androgens by liquid chromatography- tandem mass spectrometry. As DHEA was consumed, the androgen intermediates androstenediol and androstenedione were generated and subsequently converted to T. Conditioned media of DHEA- treated osteoblasts increased androgen receptor (AR) signaling, prostate- specific antigen (PSA) production, and cell numbers of the androgen- sensitive prostate cancer cell lines C4- 2B and LNCaP. DHEA did not induce AR signaling in osteoblasts despite AR expression in this cell type. We describe an unreported function of osteoblasts as a source of T that is especially relevant during androgen- responsive metastatic prostate cancer invasion into bone. © 2021 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/169292/1/jbmr4313_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/169292/2/jbmr4313.pd

CFTR expression in osteoblasts.

<p>(A) Total RNA was harvested from mouse tissues. <i>Cftr</i> mRNA expression in murine primary osteoblast cultures, pancreas, liver and lung was assessed by real-time RT PCR. (B) Calvariae were harvested from <i>Cftr</i>+/+ and <i>Cftr</i>−/− 4-day-old pups, fixed, sectioned immunostained for CFTR using the 3G11 rat anti-CFTR monoclonal antibody and counterstained with hematoxylin. (C) Osteoblasts were prepared by digestion from murine calvariae and cultured on collagen-coated glass slides. CFTR (red) was identified by immunofluorescence using the 3G11 antibody. Nuclei (blue) were identified by DAPI staining. Two adjacent osteoblasts are shown.</p

CFTR inactivation increased osteoclastogenesis and <i>Rankl</i>:<i>Opg</i> osteoblast expression.

<p>(A) Bone marrow was flushed from <i>Cftr</i>+/+ and <i>Cftr</i>−/− mice and osteoclast formation assays performed. Osteoclasts were identified by TRAP staining as cells containing ≥3 or more nuclei. More osteoclasts were found in <i>Cftr</i>−/− bone marrow. (B) <i>Rankl</i> and <i>Opg</i> mRNA expression was determined by real-time RT PCR in <i>Cftr</i>+/+ and <i>Cftr</i>−/− calvarial osteoblasts with and without PTH (1–34) 10 nM treatment for 24 hours. Overall, CFTR inactivation increased the <i>Rankl</i>:<i>Opg</i> ratio in both PTH (1–34)-treated and untreated osteoblasts. (C) In long-term osteoblast cultures, decreased <i>Opg</i> expression persisted post-confluence despite no significant difference in <i>Rankl</i>. <i>Opg</i> expression remained significantly lower at 7 days in <i>Cftr</i>−/− osteoblasts, but did not reach significance at 14 days post-confluence. (* = p<0.05; ** = p<0.01; *** = p<0.001; NS  =  not significant)</p

CFTR inactivation blocked Wnt3a and PTH-activated osteoblast canonical Wnt signaling.

<p>(A) Calvarial osteoblasts harvested from <i>Cftr</i>+/+ and <i>Cftr</i>−/− mouse pups were cultured. Cyclic AMP accumulation, a marker of PTH receptor activation, was not significantly different between <i>Cftr</i>+/+ and <i>Cftr</i>−/− osteoblasts after 10 minutes of PTH (1–34) 10 nM treatment. (B) Activation of canonical Wnt signaling was assessed utilizing the TOP-Flash and FOP-Flash Wnt luciferase reporter vectors. Canonical Wnt activity increased after 48 hours of PTH (1–34) 10 nM and Wnt3a 10 ng/ml treatment in <i>Cftr</i>+/+ but not in <i>Cftr</i>−/− calvarial osteoblasts. (* = p<0.05; NS  =  not significant)</p

CFTR inactivation reduced osteoblast differentiation and bone formation.

<p>(A) The murine calvarial organ culture assay was performed on <i>Cftr</i>+/+ and <i>Cftr</i>−/− mouse pups. After two weeks in culture, new bone formation and osteoblast number was significantly less with <i>Cftr</i> inactivation. Yellow arrows indicate the area of actively mineralizing bone indicated by orange G staining that is distinct from the red staining of eosin. (B) Murine calvarial osteoblasts harvested from <i>Cftr</i>+/+ and <i>Cftr</i>−/− calvariae were grown in culture. Seven days after reaching confluence, osteoblast differentiation was assessed by alkaline phosphatase staining. (C) Proliferation, as assessed by BrdU staining, was modestly increased in <i>Cftr</i>+/+ compared to <i>Cftr</i>−/− calvarial osteoblasts. (D) The proportion of calvarial osteoblasts that were viable, undergoing early apoptosis or late apoptosis was unchanged with CFTR inactivation. (* = p<0.05; ** = p<0.01)</p