RNA-binding protein LIN28A upregulates transcription factor HIF1α by posttranscriptional regulation via direct binding to UGAU motifs.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

The stiffness and collagen control differentiation of osteoclasts with an altered expression of c-Src in podosome.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

Endosomal TLR3 signaling in stromal osteoblasts induces prostaglandin E2-mediated inflammatory periodontal bone resorption.

Toll-like receptors (TLRs) are pattern recognition receptors that play a critical role in innate immune diseases. TLR3, which is localized in the endosomal compartments of hematopoietic immune cells, is able to recognize double-stranded RNA (dsRNA) derived from viruses and bacteria and thereby induce innate immune responses. Inflammatory periodontal bone resorption is caused by bacterial infections, which initially is regulated by innate immunity; however, the roles of TLR3 signaling in bone resorption are still not known. We examined the roles of TLR3 signaling in bone resorption using poly(I:C), a synthetic dsRNA analog. In cocultures of mouse bone marrow cells and stromal osteoblasts, poly(I:C) clearly induced osteoclast differentiation. In osteoblasts, poly(I:C) increased PGE2 production and upregulated the mRNA expression of PGE2-related genes, Ptgs2 and Ptges, as well as that of a gene related to osteoclast differentiation, Tnfsf11. In addition, we found that indomethacin (a COX-2 inhibitor) or an antagonist of the PGE2 receptor EP4 attenuated the poly(I:C)-induced PGE2 production and subsequent Tnfsf11 expression. Poly(I:C) also prolonged the survival of the mature osteoclasts associated with the increased mRNA expression of osteoclast marker genes, Nfatc1 and Ctsk. In ex vivo organ cultures of periodontal alveolar bone, poly(I:C) induced bone-resorbing activity in a dose-dependent manner, which was attenuated by the simultaneous administration of either indomethacin or an EP4 antagonist. These data suggest that TLR3 signaling in osteoblasts controls PGE2 production and induces the subsequent differentiation and survival of mature osteoclasts. Endogenous TLR3 in stromal osteoblasts and osteoclasts synergistically induces inflammatory alveolar bone resorption in periodontitis.

Prostate cancer expressing membrane-bound TGF-α induces bone formation mediated by the autocrine effect of prostaglandin E2 in osteoblasts.

Prostate cancer highly metastasizes to bone, and such cancer is associated with severe bone resorption and bone formation at the site of metastasis. Prostaglandin E2 (PGE2) promotes bone resorption in inflammatory diseases; however, the roles in prostate cancer-induced bone formation are still unclear. In the present study, we investigated the effects of membrane-bound TGF-α on prostate cancer-induced bone formation through autocrine PGE2 signaling in osteoblasts. In the prostate cancer explant experiment into tibiae, injected prostate cancer cells induced bone formation with the increased expression of osteogenic genes, such as Runx2 and Wnt5a, and prostaglandin synthase Ptgs2. In osteoblasts, PGE2 increased the number of calcified bone nodules with enhanced expression of Runx2 and Wnt5a. We also screened the factors involved in cancer progression, and 11 EGF family members were found to be expressed in the human prostate cancer cell line PC3. PC3 highly expressed amphiregulin, HB-EGF, and especially TGF-α. Treatment with recombinant TGF-α increased the Ptgs2 expression and PGE2 production in osteoblasts, which promoted the formation of calcified bone nodules, suggesting that the interaction between PC3 and osteoblasts promoted PGE2 production. In co-culture of osteoblasts and fixed PC3 cells, the phosphorylation of EGFR and ERK and subsequent Ptgs2 expression and PGE2 production were increased, an effect that was attenuated by treatment with inhibitors of EGFR and ERK. These results indicate that membrane-bound TGF-α enhances ERK signaling while also inducing PGE2-mediated bone formation in osteoblasts, thus suggesting that prostate cancer regulates both PGE2-mediated bone resorption and bone formation at the site of bone metastasis of prostate cancer.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Caveolin-3 regulates the activity of Ca2+/calmodulin-dependent protein kinase II in C2C12 cells.

Caveolins, encoded by the Cav gene family, are the main components of caveolae. Caveolin-3 (Cav3) is specifically expressed in muscle cells. Mutations in Cav3 are responsible for a group of muscle diseases called caveolinopathies, and Cav3 deficiency is associated with sarcolemmal membrane alterations, disorganization of T-tubules, and disruption of specific cell-signaling pathways. However, Cav3 overexpression increases the number of sarcolemmal caveolae and muscular dystrophy-like regenerating muscle fibers with central nuclei, suggesting that the alteration of Cav3 expression levels or localization influences muscle cell functions. Here, we used mouse C2C12 myoblasts in which Cav3 expression was suppressed with short hairpin RNA and found that Cav3 suppression impaired myotube differentiation without affecting the expression of MyoD and Myog. We also observed an increase of intracellular Ca2+ levels, total calpain activity, and Ca2+-dependent calmodulin kinase II (CaMKII) levels in Cav3-depleted myoblasts. Importantly, those phenotypes due to Cav3 suppression were caused by the ryanodine receptor activation. Furthermore, pharmacological inhibition of CaMKII rescued the impairment of myoblast differentiation due to Cav3 knockdown. Our results suggest that Cav3 regulates intracellular Ca2+ concentrations by modulating ryanodine receptor activity in muscle cells and that CaMKII suppression in muscle could be a novel therapy for caveolinopathies.

Molecular evidence of IGFBP-3 dependent and independent VD3 action and its nonlinear response on IGFBP-3 induction in prostate cancer cells.

BACKGROUND: Clinical trials have been conducted to clarify the beneficial effects of VD3 (1α,25-dihydroxy vitamin D3, also known as calcitriol) treatment in prostate cancer. However, the molecular mechanisms underlying these effects are not fully understood. Recent studies on IGFBP-3 have indicated its intracellular functions in cell growth and apoptosis. The aim of this study was to confirm the benefits of low-dose VD3 treatment and clarify the molecular mechanisms underlying these beneficial effects in prostate cancer cells. METHODS: The molecular effects of simultaneous treatment of LNCaP cells and their genetically modified cell lines with low concentration of docetaxel and VD3 were biologically and biochemically analyzed. To further determine the effects of VD3 treatment on IGFBP-3 induction system, cells were temporarily treated with VD3 in combination with a transcriptional inhibitor or protein synthesis inhibitor. Bcl-2 protein and its mRNA behavior were also observed in Igfbp-3 expression-modified LNCaP cells to determine the involvement of IGFBP-3 in the suppression of Bcl-2 by VD3 treatment. RESULTS: Changes in IGFBP-3 expression levels in LNCaP cells indicated that it mediated the inhibition of cell growth induced by VD3 treatment. IGFBP-3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancer cells to VD3 in combination with the anti-cancer drug. We further identified the distinct property of the IGFBP-3 induction system, wherein temporal VD3 stimulation-induced prolonged IGFBP-3 expression and VD3 treatment-induced increase in IGFBP-3 expression were optimized based on the protein concentration rather than the mRNA concentration. Meanwhile, Bcl-2 expression was down-regulated by VD3 treatment in an IGFBP-3-independent manner. CONCLUSION: These findings indicate the molecular mechanisms of IGFBP-3 induction stimulated by VD3 and IGFBP-3 independent Bcl-2 suppression by VD3 treatment in prostate cancer cells. The results could prompt a re-evaluation of VD3 usage in therapy for patients with prostate cancer.

Quantitative Measurements of Intercellular Adhesion Strengths between Cancer Cells with Different Malignancies Using Atomic Force Microscopy.

Intercellular adhesion strengths between two kinds of murine breast cancer cells with different malignancies were measured quantitatively using a metal cup-attached chip with atomic force microscopy (AFM). The cup-attached chip was used to approach a cell, pick it up, and then approach another cell, and the adhesion strengths were measured according to the contact time of the cells between 0 to 60 s. Separation work was used as a parameter for quantitative comparisons of the strengths. As a result, the work of a highly metastatic cancer cell (FP10SC2) was greater than a low metastatic cancer cell (4T1-LM) throughout all contact times examined. Adhesion was analyzed from a point of a view of binding kinetics of receptors on cells, and two possibilities were found: one was the number of cell adhesive receptors increased, and the other was the work to separate single molecular binding increased with increasing cancer cell malignancy. These results indicated quantitative measurements of intercellular adhesion strengths using AFM yielded information to understand the mechanism of the cancer progression from a new perspective.

Low Molecular-Weight Curdlan, (1→3)-ß-Glucan Suppresses TLR2-Induced RANKL-Dependent Bone Resorption.

Fungal ß-glucan is a potent immunological stimulator, and that it activates both the innate immune system and adaptive immunity. Curdlan is (1→3)-ß-glucan, a linear form of ß-glucan with a high molecular weight; it modulates the immune response. However, its role in bone tissue is controversial, and the effects of curdlan on bone tissues are unknown. Toll-like receptors (TLRs) play critical roles in innate immunity, and various ligands for TLRs are thought to regulate the host defense mechanisms against pathogens. TLR2 is known to form heterodimers with TLR6, and the TLR2-TLR6 heterodimer (TLR2/6) recognizes diacylated lipopeptides from Gram-positive bacteria. In the present study, we prepared low molecular-weight curdlan, (1→3)-ß-D-glucan, and examined its effects on bone resorption induced by TLR2/6 signaling. In co-cultures of bone marrow cells and osteoblasts, low molecular-weight curdlan suppressed the osteoclast formation induced by TLR2/6 ligand, and attenuated bone resorption in mouse calvarial organ cultures. Curdlan acted on mouse osteoblasts and suppressed the expression of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL), a key molecule for osteoclastogenesis. Curdlan also acted on mouse bone marrow macrophages and suppressed RANKL-dependent osteoclast differentiation from osteoclast precursor cells. The present study indicates that low molecular-weight curdlan attenuated TLR2-induced inflammatory bone resorption. Curdlan, (1→3)-ß-glucan may be a natural agent with beneficial effects on bone health in humans.

The MET/Vascular Endothelial Growth Factor Receptor (VEGFR)-targeted Tyrosine Kinase Inhibitor Also Attenuates FMS-dependent Osteoclast Differentiation and Bone Destruction Induced by Prostate Cancer.

The tyrosine kinase inhibitor TAS-115 that blocks VEGF receptor and hepatocyte growth factor receptor MET signaling exhibits antitumor properties in xenografts of human gastric carcinoma. In this study, we have evaluated the efficacy of TAS-115 in preventing prostate cancer metastasis to the bone and bone destruction using the PC3 cell line. When PC3 cells were injected into proximal tibiae in nude mouse, severe trabecular and cortical bone destruction and subsequent tumor growths were detected. Oral administration of TAS-115 almost completely inhibited both PC3-induced bone loss and PC3 cell proliferation by suppressing osteoclastic bone resorption. In an ex vivo bone organ culture, PC3 cells induced osteoclastic bone resorption when co-cultured with calvarial bone, but TAS-115 effectively suppressed the PC3-induced bone destruction. We found that macrophage colony-stimulating factor-dependent macrophage differentiation and subsequent receptor activator of NF-κB ligand-induced osteoclast formation were largely suppressed by adding TAS-115. The phosphorylation of the macrophage colony-stimulating factor receptor FMS and osteoclast related kinases such as ERK and Akt were also suppressed by the presence of TAS-115. Gene expression profiling showed that FMS expression was only seen in macrophage and in the osteoclast cell lineage. Our study indicates that tyrosine kinase signaling in host pre-osteoclasts/osteoclasts is critical for bone destruction induced by tumor cells and that targeting of MET/VEGF receptor/FMS activity makes it a promising therapeutic candidate for the treatment of prostate cancer patients with bone metastasis.