Interactions between Fibronectin and Osteoblast are Important in Osteoblast Differentiation and Mineralized Nodule Formation

Baek, Jeong-Hwa,Cho, Moo-Gil,Kim, Yong Hee,Kim, Gwan-Shik Korean Academy of Oral Biology and the UCLA Dental 2002 International Journal of Oral Biology Vol.27 No.2

Fibronectin (FN), one of bone extracellular matrix (ECM), is detected in the periosteum and the synthesis of FN is sharply increased during the early stage of osteoblast differentiation. In addition, factors known to regulate osteoblast differentiation affect both FN expression and osteoblast attachment to FN. These reports suggest that FN may play an important role in osteoblast recruitment, differentiation, and ECM organization. In the present study, we investigated the possible role of FN in osteoblast differentiation and bone formation by observing the effects of disruption of FN-osteoblast interactions. To disrupt FN-osteoblast interactions, we added to culture media 120 kDa FN fragments (120FN) which contain central cell binding domain. In addition, we observed the effect of several inhibitors of possible signaling molecules that are with the effect of FN-cell interaction disruption. 120FN reduced the expression levels of osteoblast differentiation associated genes such as alkaline phosphatase and psteocalcin and significantly suppressed mineralized nodule formation in fetal rat calvarial cell cultures. The majority of inhibitory effects seem to be occurred during early phase of osteoblast differentiation and nodule initiation, though 120 FN also delayed the nodule maturation. Among inhibitors of signaling pathways, cytochalasin B and genistein, inhibitors of actin filament rearrangement and tyrosine kinase, respectively, markedly inhibited mineralized nodule formation. Caffeic acid and aristolochic acid, inhibitors of 5-lipoxygenase and phospholipase A_2 respectively, exhibit little inhibitory effect, if any. U73122 and NPC-15437, inhibitors of phospholipase C and protein kinase C, respectively, significantly delayed nodule initiation and maturation, though the decrease in the number of mineralized nodules for at the end of culture was not that much. These findings suggest that the interactions between osteoblasts and FN may play an important role in the regulation of progressive differentiation and finally mineralized nodule formation of osteoblasts. In addition, these results raise the possibility that cytoskeleton rearrangement together with tyrosine kinase activation may be the important part of signaling from Fn-osteoblast interactions.

Dual targeting c-met and VEGFR2 in osteoblasts suppresses growth and osteolysis of prostate cancer bone metastasis

Lee, Changki,Whang, Young Mi,Campbell, Preston,Mulcrone, Patrick L.,Elefteriou, Florent,Cho, Sun Wook,Park, Serk In Elsevier 2018 Cancer letters Vol.414 No.-

<P><B>Abstract</B></P> <P>Prostate cancer characteristically induces osteoblastic bone metastasis, for which no therapies are available. A dual kinase inhibitor of c-Met and VEGFR-2 (cabozantinib) was shown to reduce prostate cancer growth in bone, with evidence for suppressing osteoblastic activity. However, c-Met and VEGFR2 signaling in osteoblasts in the context of bone metastasis remain unclear. Here we show using cultured osteoblasts that hepatocyte growth factor (HGF) and VEGF-A increased receptor activator of NFκB ligand (RANKL) and M-CSF, two essential factors for osteoclastogenesis. Insulin-like growth factor-1 (IGF1) also increased RANKL and M-CSF via c-Met transactivation. The conditioned media from IGF1-, HGF-, or VEGFA-treated osteoblasts promoted osteoclastogenesis that was reversed by inhibiting c-Met and/or VEGFR2 in osteoblasts. <I>In vivo</I> experiments used cabozantinib-resistant prostate cancer cells (PC-3 and C4-2B) to test the effects of c-Met/VEGFR2 inhibition specifically in osteoblasts. Cabozantinib (60 mg/kg, 3 weeks) suppressed tumor growth in bone and reduced expression of RANKL and M-CSF and subsequent tumor-induced osteolysis. Collectively, inhibition of c-Met and VEGFR2 in osteoblasts reduced RANKL and M-CSF expression, and associated with reduction of tumor-induced osteolysis, suggesting that c-Met and VEGFR2 are promising therapeutic targets in bone metastasis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> HGF, VEGF-A and IGF1 increase M-CSF and RANKL in osteoblasts of the bone metastasis. </LI> <LI> HGF, VEGF-A and IGF1 induce osteoclastogenesis via activation of osteoblasts. </LI> <LI> A c-Met/VEGFR2 inhibitor suppresses osteoblasts and subsequent osteoclastogenesis. </LI> <LI> Targeting c-Met and VEGFR2 in osteoblast suppresses prostate cancer bone metastasis. </LI> <LI> Osteoblasts are promising stromal cell target for the treatment of bone metastasis. </LI> </UL> </P>

형개(荊芥)가 조골세포(造骨細胞)에 미치는 영향(影響)

이주엽,황귀서,Lee, Joo-Yup,Hwang, Gwi-Seo 대한예방한의학회 2009 대한예방한의학회지 Vol.13 No.3

Objectives : The author aimed to evaluate the effect of BuOH fraction(ST) from Schizonepeta tenuifolia on osteoblast proliferation in murine calvarial cells. Methods : The osteoblast separated from murine calvariae was cultivated for 10 days and evaluated the cell function. After the addition of ST on the culture medium, we determined the effect of ST on the cell proliferation, protein synthesis, alkaline phosphatase activity, collagen synthesis, and apoptosis of the osteoblast. Results : 1. ST increased the proliferation of osteoblast, and restored the decreased cell number in glucocorticoid (GC)-treated osteoblast. 2. ST increased protein synthesis of osteoblast, and restored the decreased protein synthesis in GC-treated osteoblast. 3. ST increased ALP activity of osteoblast, and restored the decreased enzyme activity in GC-treated osteoblast. 4. ST increased collagen synthesis of osteoblast, and restored the decreased collagen synthesis in GC-treated osteoblast. 5. ST did not change the survival rate of osteoblast, but increased the survival rate in GC-treated osteoblast. Conclusions : It is concluded that ST might reduce the osteoporosis resulted from augumentation of osteoblast proliferation.

BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

Hei, Hongya,Gao, Jianjun,Dong, Jibin,Tao, Jie,Tian, Lulu,Pan, Wanma,Wang, Hongyu,Zhang, Xuemei Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.7

Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BKknockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases.

BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

Xuemei Zhang,Hongya Hei,Jianjun Gao,Jibin Dong,Jie Tao,Lulu Tian,Wanma Pan,Hongyu Wang 한국분자세포생물학회 2016 Molecules and cells Vol.39 No.7

Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BK-knockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases.

HSP27 EXPRESSION IN OSTEOBLAST BY THERMAL STRESS

임재석,김병렬,권종진,장현석,이의석,전상호,우현일,Rim, Jae-Suk,Kim, Byeong-Ryol,Kwon, Jong-Jin,Jang, Hyon-Seok,Lee, Eui-Suk,Jun, Sang-Ho,Woo, Hyeon-Il Korean Association of Maxillofacial Plastic and Re 2008 Maxillofacial Plastic Reconstructive Surgery Vol.30 No.1

Aim of the study: Thermal stress is a central determinant of osseous surgical outcomes. Interestingly, the temperatures measured during endosseous surgeries coincide with the temperatures that elicit the heat shock response of mammalian cells. The heat shock response is a coordinated biochemical response that helps to protect cells from stresses of various forms. Several protective proteins, termed heat shock proteins (hsp) are produced as part of this response. To begin to understand the role of the stress response of osteoblasts during surgical manipulation of bone, the heat shock protein response was evaluated in osteoblastic cells. Materials & methods: With primary cell culture studies and ROS 17/2.8 osteoblastic cells transfected with hsp27 encoding vectors culture studies, the thermal stress response of mammalian osteoblastic cells was evaluated by immunohistochemistry and western blot analysis. Results: Immunocytochemistry indicated that hsp27 was present in unstressed osteoblastic cells, but not fibroblastic cells. Primarily cultured osteoblasts and fibroblasts expressed the major hsp in response to thermal stress, however, the small Mr hsp, hsp27 was shown to be a constitutive product only in osteoblasts. Creation of stable transformed osteoblastic cells expressing abundant hsp27 protein was used to demonstrate that hsp27 confers stress resistance to osteoblastic cells. Conclusions: The demonstrable presence and function of hsp27 in cultured bones and cells implicates this protein as a determinant of osteoblastic cell fate in vivo.

Estradiol과 Medroxyprogesterone Acetate가 골아세포의 성장에 미치는 영향

김기석 ( Kie Suk Kim ),민부기 ( Bu Kie Min ),홍기연 ( Gi Youn Hong ),박승택 ( Seung Taek Park ),이승필 ( Seung Phil Lee ),김인숙 ( In Suk Kim ) 대한폐경학회 2001 대한폐경학회지 Vol.7 No.1

N/A This study was designed to clarify the hormonal effect on the growth of osteoblast. And the osteoblasts from neonatal mouse were cultured in the medium at various concentrations of 17β-estradiol(EST), medroxyprogesterone acetate(MPA) in addition 10nM of dexamethasone(DMS) and alcohol for 48 hours. And in order to detect the response of osteoblast on the those hormones, the alkaline phosphatase(ALP) activity also was measured. 1. At 48 hours after culture of osteoblasts in media at various concentrations of hormones, EST has an effect on increasing of the cell number in fashion of dose dependent, whereas MPA did not increase the cell number. 2. Dexametasone significantly promoted differentiation of osteoblasts does dependently with increasing of ALP activity. 3. In osteoblasts culture in media supplemented with alcohol in the absence of 10nM DMS, EST increased significantly ALP activity, while decreased ALP activity of osteoblast in the presence of DMS, 4. In culture of osteoblasts treated with alcohol in the absence of 10nM DMS, MPA did not show any change of ALP activity. But in the presence of DMS, MPA decreased ALP activity of osteoblast. 5. EST plus MPA stimulated the ALP activity in osteoblasts treated with alcohol in the absence of DMS. While, in the cells treated with DMS, they synergistically decreased ALP activity.

영지(靈芝) 추출물이 Rat fetus 두개골로부터 분리한조골세포에 미치는 영향

정은혜 ( Eun Hye Jung ),유동열 ( Dong Youl Yoo ) 대한한방부인과학회 2014 大韓韓方婦人科學會誌 Vol.27 No.2

In this study, the author aimed to evaluate the effect of EtOH extractof Ganoderma lucidum (GLE) on osteoblast proliferation in rat fetus calvarial cells. Methods: The osteoblast separated from rat fetus calvariae was cultivated for6~21 days and evaluated the cell function. After the addition of GLE on the culturemedium, we determined the effect of GLE on the cell viability, cell proliferation,bone matrix protein synthesis, alkaline phosphatase (ALP) activity, collagen synthesisand calcified nodule formation of the cultivated osteoblast. Results: GLE did not change the survival rate of rat calvarial osteoblast. GLEincreased the proliferation of rat calvarial osteoblast. GLE increased ALP activityof rat calvarial osteoblast. GLE increased bone matrix protein synthesis of ratcalvarial osteoblast. GLE increased collagen synthesis of rat calvarial osteoblast. GLE slightly affected calcified nodule formation of rat calvarial osteoblast. Conclusions: This study suggests that Ganoderma lucidum might improve theosteoporosis resulted from augmentation of osteoblast proliferation.

건지황(乾地黃) 추출물이 Rat fetus 두개골로부터 분리한 조골세포에 미치는 영향

임규정 ( Kyu Jung Im ),최경희 ( Kyung Hee Choi ),정은혜 ( Eun Hye Jung ),유동열 ( Dong Youl Yoo ) 대한한방부인과학회 2013 大韓韓方婦人科學會誌 Vol.26 No.3

Objectives: Osteoporosis is characterized by bone loss and morbidity with osteoporotic fracture. In this study, the author aimed to evaluate the effect of dried roots of Rehmannia glutinosa extract (RGE) on osteoblast proliferation in murine calvarial cells. Methods: The osteoblast separated from murine calvariae was cultivated for 6 days and evaluated the cell function. After the addition of RGE on the culture medium, we determined the effect of RGE on the cell viability, cell proliferation, protein synthesis, alkaline phosphatase activity, collagen synthesis and calcified nodule formation of the cultivated osteoblast. Results: The results were summarized as follows. 1. RGE did not change the survival rate of rat calvarial osteoblast. 2. RGE increased the proliferation of rat calvarial osteoblast. 3. RGE increased ALP activity of rat calvarial osteoblast., 4. RGE slightly affected protein synthesis of rat calvarial osteoblast. 5. RGE increased collagen synthesis of rat calvarial osteoblast. 6. RGE slightly affected calcified nodule formation of rat calvarial osteoblast. Conclusions: From these results, it is concluded that RG might improve the osteoporosis resulted from augmentation of osteoblast proliferation.

Enhancement of Osteogenic Differentiation by Combination Treatment with 5-azacytidine and Thyroid-Stimulating Hormone in Human Osteoblast Cells

선현진,송영신,조선욱,박영주 대한갑상선학회 2017 International Journal of Thyroidology Vol.10 No.2

Background and Objectives: The role of thyroid-stimulating hormone (TSH) signaling on osteoblastic differentiation is still undetermined. The aim of this study was to investigate the effects of 5-aza-2'-deoxycytidine (5-azacytidine) on TSH-mediated regulations of osteoblasts. Materials and Methods: MG63, a human osteoblastic cell-line, was treated with 5-azacytidine before inducing osteogenic differentiation using osteogenic medium (OM) containing L-ascorbic acid and β-glyceophosphate. Bovine TSH or monoclonal TSH receptor stimulating antibody (TSAb) was treated. Quantitative real-time PCR analyses or measurement of alkaline phosphatase activities were performed for evaluating osteoblastic differentiation. Results: Studies for osteogenic-related genes or alkaline phosphatase activity demonstrated that treatment of TSH or TSAb alone had no effects on osteoblastic differentiation in MG63 cells. However, treatment of 5-azacytidine, per se, significantly increased osteoblastic differentiation and combination treatment of 5-azacytidine and TSH or TSAb in the condition of OM showed further significant increase of osteoblastic differentiation. Conclusion: Stimulating TSH signaling has little effects on osteoblastic differentiation in vitro. However, in the condition of epigenetic modification using inhibitor of DNA methylation, TSH signaling positively affects osteoblastic differentiation in human osteoblasts.