Chikahisa Higuchi

Continuous Inhibition of MAPK Signaling Promotes the Early Osteoblastic Differentiation and Mineralization of the Extracellular Matrix

We screened the small molecule compounds that stimulate osteogenesis by themselves or promote bone morphogenetic protein (BMP)‐induced bone formation. We found that a specific inhibitor for MAPK/extracellular signal‐regulated kinase kinase (MEK)‐1, promoted the early osteoblastic differentiation and mineralization of extracellular matrix (ECM) in C2C12 pluripotent mesenchymal cells treated with recombinant human BMP‐2 (rhBMP‐2) and MC3T3‐E1 preosteoblastic cells. ALP activity was synergistically increased by the treatment with a specific MEK‐1 inhibitor PD98059 and rhBMP‐2 in both cell lines. Twenty‐five micromolar PD98059 promoted mineralization of ECM in rhBMP‐2‐treated C2C12 cells and MC3T3‐E1 cells. In contrast, PD98059 reduced osteocalcin (OCN) secretion and its transcriptional level in rhBMP‐2‐treated C2C12 cells but increased its secretion and mRNA level in MC3T3‐E1 cells. Stable expression of a dominant‐negative MEK‐1 mutant in C2C12 cells represented high ALP activity and low osteocalcin production in the presence of rhBMP‐2, while a constitutively active mutant of MEK‐1 attenuated both of them. Together, our results indicated that BMP‐2‐induced mineralization of ECM of pluripotent mesenchymal stem cells and preosteoblastic cells could be controlled by a fine tuning of the MAPK signaling pathway. Further, MEK‐1 inhibitors would be useful for the promotion of bone formation, for instance, the treatments for delayed fracture healing or advance of localized osteoporotic change after fracture healing.

In Vitro Generation of a Scaffold-Free Tissue-Engineered Construct (TEC) Derived from Human Synovial Mesenchymal Stem Cells: Biological and Mechanical Properties and Further Chondrogenic Potential

The purpose of this study was to characterize a tissue-engineered construct (TEC) generated with human synovial mesenchymal stem cells (MSCs). MSCs were cultured in medium with ascorbic acid 2-phosphate (Asc-2P) and were subsequently detached from the substratum. The detached cell/matrix complex spontaneously contracted to develop a basic TEC. The volume of the TEC assessed by varying initial cell density showed that it was proportional to initial cell densities up to 4 x 10(5) cells/cm(2). Assessment of the mechanical properties of TEC using a custom device showed that the load at failure and stiffness of the constructs significantly increased with time of culture in the presence of Asc-2P, while in the absence of Asc-2P, the constructs were mechanically weak. Thus, the basic TEC possesses sufficiently self-supporting mechanical properties in spite of not containing artificial scaffolding. TEC further cultured in chondrogenic media exhibited positive alcian blue staining with elevated expression of chondrogenic marker genes. Based on these findings, such human TEC may be a promising method to promote cartilage repair for future clinical application.

Comparison of Human Serum with Fetal Bovine Serum for Expansion and Differentiation of Human Synovial MSC: Potential Feasibility for Clinical Applications:

The aim of this study was to evaluate the effect of human serum (HS) on growth and differentiation capacity of human synovium-derived mesenchymal stem cells (MSC) in comparison to cells grown in fetal bovine serum (FBS). Human MSCs were isolated from the synovium of knee joints of three donors and the cells were cultured individually in varying concentrations of allogenic HS or FBS. Bovine MSCs were isolated from synovium and cultured in the same manner. Cell proliferation was assessed by the tetrazolium assay after passage 3. The capacity for chondrogenic and osteogenic differentiation was investigated in specific media followed by 1,9-dimethylmethylene blue assay and alcian blue staining, or by alizarin red staining, respectively. Human MSCs proliferated significantly more rapidly in the presence of HS than with equivalent levels of FBS. Chondrogenic or osteogenic differentiation occurred to nearly identical levels in HS or FBS. The results of this study indicate that HS is superior for the culture of human MSCs compared with FBS in terms of cellular expandability, without losing chondrogenic or osteogenic differentiation capacity. Coupled with the advantage in eliminating the potential risk accompanied with the use of xeno-derived materials, pooled, well-characterized HS could be a useful reagent to promote cellular expansion for clinical synovial stem cell-based therapy.

An Overgrowth Disorder Associated with Excessive Production of cGMP Due to a Gain-of-Function Mutation of the Natriuretic Peptide Receptor 2 Gene

We describe a three-generation family with tall stature, scoliosis and macrodactyly of the great toes and a heterozygous p.Val883Met mutation in Npr2, the gene that encodes the CNP receptor NPR2 (natriuretic peptide receptor 2). When expressed in HEK293A cells, the mutant Npr2 cDNA generated intracellular cGMP (cyclic guanosine monophosphate) in the absence of CNP ligand. In the presence of CNP, cGMP production was greater in cells that had been transfected with the mutant Npr2 cDNA compared to wild-type cDNA. Transgenic mice in which the mutant Npr2 was expressed in chondrocytes driven by the promoter and intronic enhancer of the Col11a2 gene exhibited an enhanced production of cGMP in cartilage, leading to a similar phenotype to that observed in the patients. In addition, blood cGMP concentrations were elevated in the patients. These results indicate that p.Val883Met is a constitutive active gain-of-function mutation and elevated levels of cGMP in growth plates lead to the elongation of long bones. Our findings reveal a critical role for NPR2 in skeletal growth in both humans and mice, and may provide a potential target for prevention and treatment of diseases caused by impaired production of cGMP.

IL-6 negatively regulates osteoblast differentiation through the SHP2/MEK2 and SHP2/Akt2 pathways in vitro

It has been suggested that interleukin-6 (IL-6) plays a key role in the pathogenesis of rheumatoid arthritis (RA), including osteoporosis not only in inflamed joints but also in the whole body. However, previous in vitro studies regarding the effects of IL-6 on osteoblast differentiation are inconsistent. The aim of this study was to examine the effects and signal transduction of IL-6 on osteoblast differentiation in MC3T3-E1 cells and primary murine calvarial osteoblasts. IL-6 and its soluble receptor significantly reduced alkaline phosphatase (ALP) activity, the expression of osteoblastic genes (Runx2, osterix, and osteocalcin), and mineralization in a dose-dependent manner, which indicates negative effects of IL-6 on osteoblast differentiation. Signal transduction studies demonstrated that IL-6 activated not only two major signaling pathways, SHP2/MEK/ERK and JAK/STAT3, but also the SHP2/PI3K/Akt2 signaling pathway. The negative effect of IL-6 on osteoblast differentiation was restored by inhibition of MEK as well as PI3K, while it was enhanced by inhibition of STAT3. Knockdown of MEK2 and Akt2 transfected with siRNA enhanced ALP activity and gene expression of Runx2. These results indicate that IL-6 negatively regulates osteoblast differentiation through SHP2/MEK2/ERK and SHP2/PI3K/Akt2 pathways, while affecting it positively through JAK/STAT3. Inhibition of MEK2 and Akt2 signaling in osteoblasts might be of potential use in the treatment of osteoporosis in RA.

Transient dynamic actin cytoskeletal change stimulates the osteoblastic differentiation

Dynamic cytoskeletal changes appear to be one of intracellular signals that control cell differentiation. To test this hypothesis, we examined the effects of short-term actin cytoskeletal changes on osteoblastic differentiation. We found an actin polymerization interfering reagent, cytochalasin D, promoted osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells. We also found that these effects were mediated by the protein kinase D (PKD) pathway. Short-term cytochalasin D treatment increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralization of the extracellular matrix in MC3T3-E1 cells, with temporary changes in actin cytoskeleton. Furthermore, the disruption of actin cytoskeleton induced phosphorylation of 744/748 serine within the activation loop of PKD in a dose-dependent manner. The protein kinase C (PKC)/PKD inhibitor Go6976 suppressed cytochalasin D-induced acceleration of osteoblastic differentiation, whereas Go6983, a specific inhibitor of conventional PKCs, did not. Involvement of PKD signaling was confirmed by using small interfering RNA to knock down PKD. In addition, another actin polymerization interfering reagent, latrunculin B, also stimulated ALP activity and OCN secretion with PKD activation. On the other hand, the present data suggested that transient dynamic actin cytoskeletal reorganization could be a novel cellular signal that directly stimulated osteoblastic differentiation.

Neogenin, a Receptor for Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) regulate many mammalian physiologic and pathophysiologic processes. These proteins bind with the kinase receptors BMPR-I and BMPR-II, thereby activating Smad transcription factor. In this study, we demonstrate that neogenin, a receptor for netrins and proteins of the repulsive guidance molecule family, is a receptor for BMPs and modulates Smad signal transduction. Neogenin was found to bind directly with BMP-2, BMP-4, BMP-6, and BMP-7. Knockdown of neogenin in C2C12 cells resulted in the enhancement of the BMP-2-induced processes of osteoblastic differentiation and phosphorylation of Smad1, Smad5, and Smad8. Conversely, overexpression of neogenin in C2C12 cells suppressed these processes. Our results also indicated that BMP-induced activation of RhoA was mediated by neogenin. Inhibition of RhoA promoted BMP-2-induced processes of osteoblastic differentiation and phosphorylation of Smad1/5/8. However, treatment with Y-27632, an inhibitor of Rho-associated protein kinase, did not modulate BMP-induced phosphorylation of Smad1/5/8. Taken together, our findings suggest that neogenin negatively regulates the functions of BMP and that this effect of neogenin is mediated by the activation of RhoA.

Scaffold-Free Tissue-Engineered Construct–Hydroxyapatite Composites Generated by an Alternate Soaking Process : Potential for Repair of Bone Defects

Mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC)-hydroxyapatite (HAp) composites were developed by an alternate soaking process. The TEC derived from cultured synovial MSCs was alternately immersed in varying concentrations of CaCl(2)/Tris-HCl and Na(2)HPO(4)/Tris-HCl buffers, and HAp formation was analyzed by Fourier transform infrared spectroscopy (FT-IR), wide-angle X-ray diffraction, and scanning electron microscopy (SEM). These analyses clearly demonstrated HAp formation in the TEC. Specifically, SEM assessments showed that spherical HAp crystals of approximately 1 mum were directly formed on the surfaces of the cells and extracellular matrix (ECM) fibers. Cytotoxicity from exposure to calcium or phosphate buffers of >100 mM concentrations as assessed by LIVE/DEAD staining and total DNA assays was detected, but such cytotoxicity was not detected following exposure to concentrations of <50 mM. The HAp nanocrystals (ca. approximately 500 nm) were formed after 20 cycles in 10 mM calcium or phosphate buffers, and cell survival in the composites was confirmed. Moreover, preliminary implantation of TEC-HAp composites derived from rabbit synovial MSCs to rabbit osteochondral defects exhibited accelerated osteoinduction. These composites may be the first example of a hybrid material that consists of ECM, HAp nanocrystals, and living MSCs, and the TEC-HAp composite could be a unique and useful material for bone tissue engineering.

PKCα suppresses osteoblastic differentiation

Protein kinase C (PKC) plays an essential role in cellular signal transduction for mediating a variety of biological functions. There are 11 PKC isoforms and these isoforms are believed to play distinct roles in cells. Although the role of individual isoforms of PKC has been investigated in many fields, little is known about the role of PKC in osteoblastic differentiation. Here, we investigated which isoforms of PKC are involved in osteoblastic differentiation of the mouse preosteoblastic cell line MC3T3-E1. Treatment with Gö6976, an inhibitor of PKCα and PKCβI, increased alkaline phosphatase (ALP) activity as well as gene expression of ALP and Osteocalcin (OCN), and enhanced calcification of the extracellular matrix. Concurrently, osteoblastic cell proliferation decreased at a concentration of 1.0 μM. In contrast, a PKCβ inhibitor, which inhibits PKCβI and PKCβII, did not significantly affect osteoblastic differentiation or cell proliferation. Knockdown of PKCα using MC3T3-E1 cells transfected with siRNA also induced an increase in ALP activity and in gene expression of ALP and OCN. In contrast, overexpression of wild-type PKCα decreased ALP activity and attenuated osteoblastic differentiation markers including ALP and OCN, but promoted cell proliferation. Taken together, our results indicate that PKCα suppresses osteoblastic differentiation, but promotes osteoblastic cell proliferation. These results imply that PKCα may have a pivotal role in cell signaling that modulates the differentiation and proliferation of osteoblasts.

Inorganic polyphosphate differentiates human mesenchymal stem cells into osteoblastic cells

The existence of inorganic polyphosphates [poly(P)] in human cells has been demonstrated. In osteoblasts, it is suggested that the concentration of cellular poly(P) is relatively high. In this study, we examined whether poly(P) accelerates the differentiation of human mesenchymal stem cells (hMSCs) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) into osteoblastic cells. Alkaline phosphatase (ALP) activity was induced by poly(P) in hMSCs from both OA and RA. In Alizarin Red S and osteocalcin EIA, there was a significant difference between the control and poly(P) group. In real-time PCR, there was a significant difference in ALP, collagen type 1A, osteocalcin, and bone sialoprotein between the control and poly(P) group. Our findings suggest that poly(P) have the potent role of differentiating hMSCs into osteoblastic cells at the early and later stages of osteoblastic differentiation.