Decreased Alkaline Phosphatase Activity Research Articles

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.

The Mechanism of Cordyceps sinensis and Strontium in Prevention of Osteoporosis in Rats

The effects of Cordyceps sinensis (Caterpillar fungus) and strontium ranelate on ovariectomized osteopenic rats was studied in this paper. After the rats were treated orally with C. sinensis, strontium, and C. sinensis rich in strontium ranelate (CSS) respectively, serum alkaline phosphatase (ALP), tartarate-resistant acid phosphatase (TRAP), serum osteocalcin (OC), homocysteine, C-terminal crosslinked telopeptides of collagen type I (CTX), estradiol, and interferon-gamma (IFN-γ) level were examined. The beneficial effects of CSS on improvement of osteoporosis in rats were attributable mainly to decrease ALP activity, TRAP activity, CTX level, and IFN-γ level. At the same time, CSS also increase the OC and estradiol level in ovariectomized osteopenic rats. This study demonstrates the value of C. sinensis rich in strontium ranelate in the management of postmenopausal osteoporosis in humans.

Activity of Soil Dehydrogenases, Urease, and Acid and Alkaline Phosphatases in Soil Polluted with Petroleum

This study was undertaken to (1) determine the effects of petroleum pollution on changes in the biochemical properties of soil and (2) demonstrate whether the application of compost, bentonite, and calcium oxide is likely to restore biological balance. Petroleum soil pollution at a dose ranging from 2.5 to 10 cm3/kg disturbed the biochemical balance as evidenced by inhibition of the activities of soil dehydrogenases (SDH), urease (URE), and acid phosphatase (ACP). The greatest change was noted in the activity of SDH, whereas the least change occurred in URE. Petroleum significantly increased the activity of soil alkaline phosphatase (ALP) in soil used for spring rape, whereas in soil used for oat harvest there was decreased ALP activity. The application of compost, bentonite, and calcium oxide to soil proved effective in mitigating the adverse effects of petroleum on the activities of soil enzymes. Soil enrichment with compost, bentonite, and calcium oxide was found to stimulate the activities of URE and ALP and inhibit the activity of ACP. The influence of bentonite and calcium oxide was greater than that of compost. Calcium oxide and, to a lesser extent, compost were found to increase the activity of SDH, whereas bentonite exerted the opposite effect, especially in the case of the main crop, spring rape. The activities of SDH, URE, and ACP were higher in soil used for rape than that for oats. In contrast the activity of ALP was higher in soil used for oats. Data thus indicate that compost and especially bentonite and calcium oxide exerted a positive effect on activities of some enzymes in soil polluted with petroleum. Application of neutralizing additives to soil restored soil biological balance by counteracting the negative influence of petroleum on activities of URE and ALP.

Aging affects the phenotypic characteristics of human periodontal ligament cells and the cellular response to hormonal stimulation in vitro

Aging modulates the proliferative activity and organic matrix production of cells in vivo and in vitro. Here, we explore how aging affects the phenotypic characteristics of human periodontal ligament cells and their response to hormonal stimulation. Fifth passage periodontal ligament cells from subjects aged 12-14 (group 1), 41-55 (group 2) and 61-70 years (group 3) were characterized for the expression of mesenchymal marker genes and proteins by real-time PCR and flow cytometry. Confluent cultures were exposed to 10(-12) m parathyroid hormone(1-34) [PTH(1-34)] intermittently for three cycles. At harvest, cell number, alkaline phosphatase activity and osteocalcin production were determined by cell count, biochemical assay and ELISA. The characterization of the cells revealed a decreased expression of osteoblast-specific marker genes along with a lower percentage of cells presenting the respective proteins with age. An intermittent exposure of the cultures to 10(-12) m PTH(1-34) induced an increase of the cell number as opposed to a significant decrease of alkaline phosphatase activity and osteocalcin production. The cellular response to PTH(1-34) was strongest in group 1. Basal osteoprotegerin levels were highest in the cultures from the oldest donors and inhibited by intermittent PTH(1-34) in all groups. Our data indicate that periodontal ligament cells from older subjects display a less differentiated phenotype and a reduced response to intermittent PTH, suggesting a compromised ability to maintain tissue homeostasis and a limited possibility to support periodontal repair processes with age. The high basal osteoprotegerin expression in older subjects might serve as a compensatory mechanism.

Functional expression of double‐stranded RNA‐dependent protein kinase in rat intestinal epithelial cells

Intestinal epithelial cells (IECs) are exposed to external environment, microbial and viral products, and serve as essential barriers to antigens. Recent studies have shown that IECs express Toll-like receptors (TLRs) and respond to microbial components. The antimicrobial and antiviral barriers consist of many molecules including TLRs. To investigate the further component of this barrier in intestine, we examined the expression of double-stranded RNA-dependent protein kinase (PKR). PKR is a player in the cellular antiviral response and phosphorylates alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha) to block protein synthesis and induces apoptosis. In this study, we showed that the expression of PKR was restricted to the cytoplasm of absorptive epithelial cells in the intestine of adult rat. We also demonstrated that PKR was expressed in the cultured rat intestinal epithelial cells (IEC-6). The level of PKR protein expression and the activity of alkaline phosphatase (ALP) increased in the cultured IEC-6 cells in a time-dependent manner. Inhibition of PKR by the 2-aminopurine treatment decreased ALP activity in the IEC-6 cells. Treatment of IEC-6 cells with synthetic double-stranded RNA (dsRNA) induced cell death in a dose-dependent manner. The addition of hydrocortisone also provoked suppression of PKR expression and ALP activity. This modulation might be mediated by signal transducers and activators of transcription-1 (STAT-1) protein. We concluded that PKR is expressed in IECs as potent barriers to antigens and is a possible modulator of the differentiation of rat IECs.

Disease-Associated Extracellular Matrix Suppresses Osteoblastic Differentiation of Human Periodontal Ligament Cells Via MMP-1

Fibronectin (FN) fragments found in chronic inflammatory diseases, including periodontal disease and arthritis, may contribute to tissue destruction in part via induction of matrix metalloproteinases (MMPs). We previously showed that the 120-kDa FN fragment containing the central cell binding domain (120FN) dose dependently induces MMP-1 (collagenase-1) in human periodontal ligament (PDL) cells, whereas intact FN did not elicit this response. Recently, we found that an increase in MMP-1 expression is accompanied by a decreased osteoblastic phenotype in PDL cells. We hypothesized that 120FN inhibits osteoblastic differentiation of PDL cells by inducing MMP-1. Effects of increasing concentrations of 120FN on MMP-1 expression and on osteoblastic markers were assessed in cultured PDL cells using Western blotting, qRT-PCR, and collagen degradation and alkaline phosphatase (AP) activity assays. The 120FN dose dependently increased MMP-1 expression and activity, concomitant with a decrease in AP activity. The increase in collagenase activity was largely attributed to increased MMP-1 expression. Concurrent with the decrease in AP activity, the 120FN reduced baseline and dexamethasone-induced gene expression of specific osteoblastic markers, Runx2 and osteonectin, and diminished mineralized nodule formation. Finally, siRNA inhibition of 120FN-induced MMP-1 reduced collagenase expression and rescued the AP phenotype to baseline levels. These findings suggest that disease-associated 120FN, in addition to having direct effects on tissue destruction by upregulating MMPs, could contribute to disease progression by impeding osteoblastic differentiation of osteogenic PDL cells and, consequently, diminish bone regeneration.

Effect of Short-Time Povidone-Iodine Application on Osteoblast Proliferation and Differentiation

Background and Objective:Povidone-iodine [polyvinylpyrrolidone-iodine complex (PVP-I)] is a broad-spectrum antimicrobial agent, frequently used in dentistry. In this study we investigated the short- and longterm effects on osteoblast number, viability, and function after short exposure to PVP-I with and without additional bone-morphogenetic protein-2 (BMP-2). Material and Methods:Confluent osteoblast-like cell line (MC3T3-E1, subclone 24) cultures were exposed to pure PVP-I solution (7.7 mg/ml) and dilutions of 1:10, 1:100 and 1:1000 for 10 seconds and washed with phosphate buffer solution. Cell proliferation and viability was determined by MTT and differentiation status by alkaline phosphatase (ALP) activity 6 days after initial plating. In a separate experiment, long-term cell proliferation, viability and function were assessed 4 weeks after PVP-I treatment by MTT and deposited calcium using an Alizarin-red staining test.Results:PVP-I decreased ALP activity substantially. Stimulation by BMP-2 recovered ALP activity to near control levels at 1:100 and 1:1000 dilutions of PVP-I. The MTT assay showed reduced proliferation of the preosteoblastic cells for all treatments, irrespective whether BMP-2 was used or not. Only at PVP-I dilutions of 1:1000 proliferation rate was back to normal levels (95.6±2.4 %). No adverse long-term effect of PVP-I on mineralization of the extracellular matrix (Alizarinred) for dilutions higher than 1:100 was observed. Interestingly, undiluted and 1:10 diluted PVP-I even showed a significant increase in mineral deposition, especially in the presence of BMP-2.Conclusion:Short-time application of PVP-I in concentrations of 1:10 and higher lead to decreased viability and impaired differentiation. However, surviving cells showed good recovery and mineralization potential.

Effects of transgenic Pit-1 overexpression on calcium phosphate and bone metabolism

The type III inorganic phosphate (Pi) transporter Pit-1 was previously found to be preferentially expressed in developing long bones. Several studies also described a regulation of its expression in cultured bone cells by osteotropic factors, suggesting a role of this transporter in bone metabolism. In the present study, we investigated the effects of the transgenic overexpression of Pit-1 in Wistar male rats on calcium phosphate and bone metabolism. A threefold increase and doubling of Pi transport activity were recorded in primary cultured osteoblastic cells derived from calvaria of two transgenic (Tg) lines compared with wild-type littermates (WT), respectively. Skeletal development was not affected by the transgene, and bone mass, analyzed by DXA, was slightly decreased in Tg compared with WT. Enhanced Pi uptake in calvaria-derived osteoblasts from Pit-1 Tg was associated with a significantly decreased expression of alkaline phosphatase activity and a normal deposition and calcification of the collagenous matrix. In 4-month-old adult Tg rats, serum Pi and renal Pi transport were increased compared with WT. The decrease of serum Ca concentration was associated with increased serum parathyroid hormone levels. Variations in serum Pi in Pit-1 Tg rats were negatively correlated with serum fibroblast growth factor-23, whereas 1,25-dihydroxyvitamin D(3) was not affected by Pit-1 overexpression. In conclusion, transgenic Pit-1 overexpression in rats affected bone and calcium phosphate metabolism. It also decreased alkaline phosphatase activity in osteoblasts without influencing bone matrix mineralization as well as skeletal development.

Cortistatin attenuates vascular calcification in rats

Cortistatin (CST) is a newly discovered polypeptide with multiple biological activities that plays a regulatory role in the nervous, endocrine and immune systems. However, the role of CST in the pathogenesis of cardiovascular diseases remains unclear. In this study, we investigated in rats whether CST inhibits vascular calcification induced by vitamin D3 and nicotine treatment in vivo and calcification of cultured rat vascular smooth muscular cells (VSMCs) induced by beta-glycerophosphate in vitro and the underlying mechanism. We measured rat hemodynamic variables, alkaline phosphatase (ALP) activity, calcium deposition and pathological changes in aortic tissues and cultured VSMCs. CST treatment significantly improved hemodynamic values and arterial compliance in rats with vascular calcification, by decreasing systolic blood pressure, pulse pressure, left ventricular end-systolic pressure and left ventricular end-diastolic pressure. CST also significantly decreased ALP activity and calcium deposition, alleviated pathological injury and down-regulated the mRNA expression of type III sodium-dependent phosphate co-transporter-1 (Pit-1) in aortic tissues. It dose-independently inhibited the calcification of VSMCs by decreasing ALP activity and calcium deposition, alleviating pathologic injury and down-regulating Pit-1 mRNA expression. As with CST treatment, ALP activation and calcium deposition were decreased significantly on treatment with ghrelin, the endogenous agonist of growth hormone secretagogue receptor 1a (GHSR1a), but not significantly with somatostatin-14 or proadrenomedullin N-terminal 20 peptide in VSMCs. Further, growth hormone-releasing peptide-6[D-lys], the endogenous antagonist of GHSR1a, markedly reversed the increased ALP activity and calcium deposition in VSMCs. CST could be a new target molecule for the prevention and therapy of vascular calcification, whose effects are mediated by GHSR1a rather than SSTRs or Mrg X2.

Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression.

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generated mice lacking the Galnt3 gene, which developed hyperphosphatemia without apparent calcifications. In response to hyperphosphatemia, Galnt3-deficient mice had markedly increased Fgf23 expression in bone. However, compared with wild-type and heterozygous littermates, homozygous mice had only about half of circulating intact Fgf23 levels and higher levels of C-terminal Fgf23 fragments in bone. Galnt3-deficient mice also exhibited an inappropriately normal 1,25-dihydroxyvitamin D level and decreased alkaline phosphatase activity. Furthermore, renal expression of sodium-phosphate cotransporters and Kl were elevated in Galnt3-deficient mice. Interestingly, there were sex-specific phenotypes; only Galnt3-deficient males showed growth retardation, infertility, and significantly increased bone mineral density. In summary, ablation of Galnt3 impaired secretion of intact Fgf23, leading to decreased circulating Fgf23 and hyperphosphatemia, despite increased Fgf23 expression. Our findings indicate that Galnt3-deficient mice have a biochemical phenotype of tumoral calcinosis and provide in vivo evidence that Galnt3 plays an essential role in proper secretion of Fgf23 in mice.

Low magnitude and high frequency mechanical loading prevents decreased bone formation responses of 2T3 preosteoblasts

Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the random positioning machine (RPM) and intervened with an LMHF mechanical load (0.1-0.4 g at 30 Hz for 10-60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20 ng/ml). However, LMHF mechanical loading prevented the RPM-induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM-induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse-induced bone loss.

Effects of granulocyte colony-stimulating factor and stem cell factor, alone and in combination, on the biological behaviours of bone marrow mesenchymal stem cells

Aim: The effects of granulocyte colony- stimu-lating factor (G-CSF) and stem cell factor (SCF) on the proliferation and osteogenic differentia-tion capacity of bone marrow mesenchymal stem cells (MSCs) were studied in the experi-ment. Methods: Bone marrow MSCs were col-lected from rabbits successfully, and treated with various concentrations of G-CSF, SCF or a combination of the two. Flow cytometric ana-lyse, MTT test, CFU-F assay, and alkaline phosphatase (ALP) activity measurement were employed. Results: The results of flow cytome-try showed that immunophenotype of the cells were CD29+/CD45-, CD105+/ CD34–, CD90+/ HLADR–. MSCs were shown to constitutively express low levels of c-kit which could be en-hanced by SCF. G-CSF and SCF had an obvious facilitative effect on the proliferation of MSCs in a dose-dependent fashion. In addition, G-CSF and SCF would be effective in reversibly pre-venting their differentiation, as showed by the decrease of ALP activity, leading to self-renewal rather than differentiative cell divisions. The effects of G-CSF were superior to SCF. And cells in the group treated with combination of G-CSF and SCF showed more powerful effects than the groups treated with G-CS, SCF, or none of the two. Conclusion: On the whole, these studies demonstrated that MSCs responsed to G-CSF, SCF, and to G-CSF plus SCF in a manner that suppressed differentiation, and promotes proliferation and self-renewal, and support the view that these factors could act synergistically.

Effect of Bone Morphogenetic Protein-2 (BMP-2) or Troglitazone, as an Inducer of Osteogenic Cells or Adipocytes, on Differentiation of a Bone Marrow Mesenchymal Progenitor Cell Line Established from Temperature-Sensitive (ts) Simian Virus (SV) 40 T-Antigen Gene Transgenic Mice

TBR31-2 is one of the bone marrow stromal cell lines. Differentiation toward osteogenic cells and calcification was observed when TBR31-2 cells were cultured for 4 weeks. Bone morphogenetic protein-2 (BMP-2) stimulated alkaline phosphatase (ALP) activity in a dose- and time-dependent manner. On the other hand, troglitazone increased oil droplet accumulation in a dose-dependent manner. In the presence of BMP-2, an increase of expression in osteogenic cell differentiation marker genes and a decrease of expression in adipocyte differentiation marker genes were observed with the exception of the induced expression of peroxisome proliferator-activated receptor gamma (PPARgamma), however, troglitazone, a ligand of PPARgamma treatment exhibited the opposite tendency. Interestingly, treatment with both BMP-2 and troglitazone resulted in a decrease of ALP activity and an increase of oil droplet accumulation. Reverse tanscription-polymerase chain reaction (RT-PCR) analysis also indicated that osteogenic differentiation markers decreased and that adipocyte differentiation markers increased. Thus, when the cells were cultured with BMP-2, osteogenic differentiation was enhanced while the expression of PPARgamma was maintained, and the addition of troglitazone caused a significant number of differentiated cells into adipocytes. These findings indicate that BMP-2 enhanced osteogenic differentiation and the expression of adipogenic transcription factor (PPARgamma) followed by osteogenic differentiation without activation of PPARgamma by its ligand.

Advanced Oxidation Protein Products Inhibit Proliferation and Differentiation of Rat Osteoblast-like Cells via NF-κB Pathway

Background/Aims: Advanced oxidation protein products (AOPPs), a novel marker for oxidative protein damage, regulated cell behavior in many cell types. Although previous reports have revealed that oxidative stress resulted in an increased AOPPs content in cultured osteoblast-like cells, the effects of AOPPs on osteoblast-like cell function remain unclear. This study aimed to investigate the effect of AOPPs on the proliferation and differentiation of rat osteoblast-like (ROB) cells in vitro. Methods: ROB cells isolated from calvarias of newborn rats were incubated with AOPPs-modified rat serum albumin (AOPPs-RSA) prepared in vitro by incubation of RSA with hypochlorous acid. Cell proliferation was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Alkaline phosphatase activity was measured. Expression of osteocalcin mRNA and protein were examined using reverse transcriptase-polymerase chain reaction and Western blot, respectively. Reactive oxygen species (ROS) generation was detected by flow cytometry and fluorescent microscope. Phosphorylation of nuclear factor (NF)- κB p65 subunit was detected by Western blot. Results: Exposure of ROB cells to AOPPs-RSA significantly inhibited cell proliferation, decreased alkaline phosphatase activity, down-regulated the expression of osteocalcin mRNA and protein in both a concentration- and time-dependent manner. AOPPs challenge induced ROS generation and NF-κB p65 phosphorylation, which were inhibited by superoxide dismutase, catalase and NADPH oxidase inhibitors diphenyleneiodonium and apocynin. Furthermore, NF-κB inhibitor SN50 could reverse the AOPPs-induced inhibition of ROB cell proliferation and differentiation. Conclusion: These results suggest that AOPPs can inhibit proliferation and differentiation of ROB cells through ROS-dependent NF-κB pathway.

Cissus quadrangularis extract enhances biomineralization through up-regulation of MAPK-dependent alkaline phosphatase activity in osteoblasts

Cissus quadrangularis Linn. has been implicated as therapeutic agent for enhancing bone healing. Though its osteogenic activity has been suggested, the underlying mechanism still remains unclear. In the present study, the effects of ethanol extract of C. quadrangularis (CQ-E) on osteoblast differentiation and function were analyzed using murine osteoblastic cells. The results indicated that mRNA expressions of osteoblast-related genes were not affected by the CQ-E treatment. However, alkaline phosphatase (ALP) activity and the extent of mineralized nodules were significantly increased in treated cells compared with controls. The addition of an extracellular regulated kinase 1/2 inhibitor, a Jun N-terminal kinase 1/2/3 inhibitor and a p38 mitogen-activated protein kinase (MAPK) inhibitor resulted in significantly decreased ALP activity, preferentially by p38 MAPK inhibitor. These results suggested that CQ-E may regulate osteoblastic activity by enhancing ALP activity and mineralization process, and the increased ALP activity effect of CQ-E is likely mediated by MAPK-dependent pathway.

Annexin-Mediated Matrix Vesicle Calcification in Vascular Smooth Muscle Cells

In bone, osteoblasts and chondrocytes synthesize matrix vesicles (MVs) that interact with collagen to initiate calcification. MVs have been identified in human calcified arteries but are poorly characterized. The objective of this study is to determine the role of annexins and fetuin-A in MV formation and activity during calcification in bovine vascular smooth muscle cells (BVSMCs). BVSMCs were treated with control or calcification (high phosphorus) media, and cellular MVs were isolated by collagenase digestion and secreted MVs were isolated from cultured media by ultracentrifugation. The results showed that alkaline phosphatase (ALP) activity was significantly increased in MVs from calcified BVSMCs compared with noncalcified BVSMCs, as was annexin II and VI content and (45)Ca uptake. We also determined that MVs from calcifying BVSMCs could mineralize type I collagen but not type II collagen in the absence of cells in a dose- and time-dependent manner. Blockade of annexin calcium channel activity by K201 significantly decreased ALP activity and reduced the ability of the MVs to subsequently calcify on collagen, whether the K201 was added during or after MV formation. Furthermore, cellular MVs had significantly increased ability to calcify on collagen compared with secreted MVs, likely because of their increased ALP activity and annexin II content but low fetuin-A content. In conclusion, our results suggest that mineralization in VSMCs requires both active MVs and an interaction of the MVs with type I collagen, and both steps require annexin activity.

Dihydroxy-cholecalciferol stimulates adipocytic differentiation of porcine mesenchymal stem cells

Dihydroxy-cholecalciferol [1,25(OH) 2D 3] has been shown to have pleiotropic effects on the differentiation of mesenchymal stem cells (MSC) based on species and culture conditions. We have examined the effects of 1,25(OH) 2D 3 on the differentiation of porcine MSC under culture conditions designed to promote proliferation in order to attempt to mimic the conditions in young, rapidly growing animals. The MSC were isolated from bone marrow of a young pig and grown in basal media (BM) containing DMEM+10% fetal bovine serum and antibiotics. Cells received either BM, BM+10 −8 M 1,25(OH) 2D 3 or BM+10 −7 M 1,25(OH) 2D 3 with complete media changes every 3 days for a total of 12 days of culture. On days 3, 6, 9 and 12, viable cell numbers were determined, and samples were collected for gene expression analysis and cytochemical staining. There was a treatment-based reduction in cell numbers on 6, 9 and 12 days ( P<.05). The concentrations of mRNAs encoding peroxisome proliferator-activated receptor gamma, lipoprotein lipase, and adipocyte-binding protein 2 were increased ( P<.05) in a manner indicative of adipocytic differentiation by treatment with 1,25(OH) 2D 3 in a dose-dependent manner. However, the mRNA levels of osteocalcin, a late stage marker of osteoblastic differentiation, was also increased ( P<.05) by treatment with 1,25(OH) 2D 3. An increased percentage of lipid filling, based on Oil Red O staining, and decreased alkaline phosphatase activity, was also seen with 1,25(OH) 2D 3 treatment. These data suggest that 1,25(OH) 2D 3 stimulates the differentiation of porcine MSC towards an adipocytic phenotype.

PDGF Receptor β Is a Potent Regulator of Mesenchymal Stromal Cell Function

Mesenchymal stromal cells (MSCs) in bone marrow are important for bone homeostasis. Although platelet-derived growth factor (PDGF) has been reported to be involved in osteogenic differentiation of MSCs, the role remains controversial and the network of PDGF signaling for MSCs has not been clarified. To clarify the underlying regulatory mechanism of MSC functions mediated by PDGF, we deleted the PDGF receptor (PDGFR)beta gene by Cre-loxP strategy and examined the role of PDGF in osteogenic differentiation of MSCs and fracture repair. In cultured MSCs, the mRNA expression of PDGF-A, -B, -C, and -D as well as PDGFRalpha and beta was detected. Depletion of PDGFRbeta in MSCs decreased the mitogenic and migratory responses and enhanced osteogenic differentiation as evaluated by increased alkaline phosphatase (ALP) activity and mRNA levels of ALP, osteocalcin (OCN), bone morphogenetic protein (BMP) 2, Runx2, and osterix in quantitative RT-PCR. PDGF-BB, but not PDGF-AA, inhibited osteogenic differentiation accompanied by decreased ALP activity and mRNA levels, except for BMP2. These effects of PDGF-BB were eliminated by depletion of PDGFRbeta in MSCs except that PDGF-BB still suppressed osterix expression in PDGFRbeta-depleted MSCs. Depletion of PDGFRbeta significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFRbeta gene deletion, we showed that PDGFRbeta signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFRalpha on the osteogenic differentiation were very subtle. PDGFRbeta could represent an important target for guided tissue regeneration or tissue engineering of bone.

Oxygen tension regulates preosteocyte maturation and mineralization

Oxygen availability is a critical signal for proper development of many tissues, however there is limited knowledge of its role in the maturation of bone cells. To test the hypothesis that low pO2 regulates bone cell mineralization, MLO-A5 and MLO-Y4 cells were cultured in monolayer and three-dimensional alginate scaffolds in hypoxia (2% O2) or normoxia (20% O2). Hypoxia reduced mineralization and decreased alkaline phosphatase activity of preosteocyte-like MLO-A5 cells in both monolayer and alginate cultures. Similar changes in osteogenic activity were seen when the were subjected to chemical hypoxia. Likewise, Osteocyte-like MLO-Y4 cells also exhibited reduced osteogenic activity in hypoxia relative to normoxic controls. Based on these observations, it is concluded that a low pO2 decreased the mineralization potential of bone cells at both early and late stages of maturation. Since the oxemic state is transduced by the transcription factor, HIF-1α, experiments were performed to determine if this protein was responsible for the observed changes in mineral formation. It was noted that when HIF-1α was silenced, mineralization activities were not restored. Indeed, in hypoxia, in relationship to wild type controls, the mineralization potential of the knockdown cells was further reduced. Based on these findings, it is concluded that the osteogenic activity of preosteocyte-like cells is dependent on both the O2 tension and the expression of HIF-1α.

Wdr5 Is Essential for Osteoblast Differentiation

Wdr5 is developmentally expressed in osteoblasts and accelerates osteoblast differentiation in vitro and in vivo. To address whether Wdr5 is essential for osteoblast differentiation, plasmid-based small interfering RNAs were used to stably suppress endogenous Wdr5 protein levels in MC3T3-E1 cells. Reduction of endogenous Wdr5 levels markedly inhibited osteoblast differentiation, evidenced by a significant decrease in alkaline phosphatase activity, Runx-2 and osteocalcin mRNAs, and absence of mineralized matrix formation. Wdr5 suppression also resulted in a reduction of histone H3 lysine 4 trimethylation, confirming its critical role in this modification. Because Wdr5 overexpression enhances canonical Wnt signaling in osteoblasts in vivo, the effects of Wdr5 silencing on this pathway were examined. The expression of the canonical Wnt target gene, c-myc, was decreased, whereas that of sfrp2, which is repressed by Wnt signaling, was increased with Wdr5 knockdown. Although only a minimal increase in apoptosis was observed, the antiapoptotic effect of Wnt signaling was also impaired with Wdr5 silencing. The expression of canonical Wnts was significantly decreased with Wdr5 knockdown, resulting in a decrease in nuclear beta-catenin protein levels. Activation of the canonical Wnt signaling pathway did not overcome the effects of Wdr5 knockdown on the expression of Wnt target genes. Chromatin immunoprecipitation demonstrated that Wdr5 is present on the Wnt1 promoter and on canonical Wnt response elements of the c-myc and Runx-2 promoters. These studies demonstrate that Wdr5 suppression interferes with the canonical Wnt signaling pathway at multiple stages and that optimal Wdr5 levels are required for induction of the osteoblast phenotype.