Loss of Lgr4 inhibits differentiation,migration and apoptosis,and promotes proliferation in bone mesenchymal stem cells

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4^+/– mice at E16.5 by β-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4^−/−) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4^−/−) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy.     

Serum inhibition of proliferation of serum-free mouse embryo cells

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominantly diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.     

CTRP3/cartducin promotes proliferation and migration of endothelial cells

Nicotinic acid (niacin) has been used clinically to manage dyslipidemia for many years. The molecular target of nicotinic acid was unknown until the recent revelation of human G-coupled receptor HM74a as the high affinity receptor for nicotinic acid. In searching for a cell line expressing endogenous human HM74a receptor, we have identified that the A431 cell line, a human epidermoid cell line, expresses a high level of HM74a receptor. An HM74a-specific real time PCR probe set was designed and the mRNA levels of HM74a in A431 and 32 other cultured cell lines were measured quantitatively. When the mRNA expression of HM74a in A431 cells was compared to that in human primary preadipocytes, adipocytes and adipose tissue, we found that the level in A431 was about 10- fold higher than that in adipocytes and adipose tissue. The ratio of HM74a:HM74 mRNA was measured quantitatively and it was determined to be 3:2 in A431 cells. The function of the HM74a receptor in A431 cells was evaluated for its ability to inhibit forskolin-induced cAMP production. Pertussis toxin treatment abolished the inhibition. Our data suggest that the A431 cell line may serve as a cellular model for further investigation of niacin/HM74a-mediated signal transduction in modulating metabolism. A431 cell line may also provide a valuable cell model to study prostaglandin production upon HM74a activation to improve our understanding of niacin/HM74a-mediated skin flushing. The first two author contributed equally.     

Hyaluronic acid promotes chick embryo fibroblast and chondroblast expression

15-day-chick-embryo fibroblasts and chondroblasts were cultured in the presence of high and low molecular weight exogenous hyaluronic acid (HA). Growth range and incorporation of radiolabelled sulphate and proline were determined. HA reduced cell proliferation to about 75% of controls, while incorporation of radiolabelled sulphate and proline was higher in HA-treated cultures of both chondroblasts and fibroblasts. The effect was not due to the polyanionic or polymeric nature of the molecule and appeared to be highly specific for HA.     

Insulin-like growth factor-I promotes proliferation and migration of cavernous smooth muscle cells

To better understand the physiology of cavernous smooth muscle cells (CSMC), particularly their regulation by IGF-I, we isolated CSMC from rats of various ages and grew them as cell cultures. CSMC from very young (1 week of age) and very old (28 months of age) rats secreted the least amounts of IGF-I, and those from 16-week-old rats the most. IGF-I stimulated growth of CSMC at an optimal concentration of 12.5 ng/ml. At this concentration, CSMC from 11-week-old rats showed the highest growth rate and CSMC from 28-month-old rats showed the lowest. The optimal IGF-I concentration for migration of CSMC was 10 ng/ml. At this concentration, CSMC from 4-week-old rats showed the highest migratory rate and CSMC from 28-month-old rats showed the lowest. IGF-I also stimulated VEGF secretion from CSMC at an optimal concentration of 10 ng/ml. At this concentration, CSMC from 16-week-old rats secreted VEGF the most and CSMC from 28-month-old rats secreted the least. The expression levels of IGF-IR paralleled the IGF-I-regulated growth rates of these cells. Expression of IGF-IR was identified in the cavernous smooth muscle and the urethra epithelium of the penis.     

Thymidine kinase activity in mouse embryo fibroblast cells infected with murine cytomegalovirus

Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-³H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication.     

Spondin 2 promotes the proliferation,migration and invasion of gastric cancer cells

Spondin 2 (SPON2), a member of the Mindin F‐Spondin family, identifies pathogens, activates congenital immunity and promotes the growth and adhesion of neurons as well as binding to their receptors, but its role in promoting or inhibiting tumour metastasis is controversial. Here, we investigated its expression levels and mechanism of action in gastric cancer (GC). Western blotting and GC tissue arrays were used to determine the expression levels of SPON2. ELISAs were performed to measure the serum levels of SPON2 in patients with GC. Two GC cell lines expressing low levels of SPON2 were used to analyse the effects of regulating SPON2 expression on proliferation, migration, invasion, the cell cycle and apoptosis. The results revealed that SPON2 was highly expressed in GC tissues from patients with relapse or metastasis. The levels of SPON2 in sera of patients with GC were significantly higher compared with those of healthy individuals and patients with atrophic gastritis. Knockdown of SPON2 expression significantly inhibited the proliferation, migration and invasion of GC cells in vitro and in vivo. Down‐regulation of SPON2 arrested the cell cycle in G1/S, accelerated apoptosis through the mitochondrial pathway and inhibited the epithelial‐mesenchymal transition by blocking activation of the ERK1/2 pathway. In summary, this study suggests that SPON2 acts as an oncogene in the development of GC and may serve as a marker for the diagnosing GC as well as a new therapeutic target for GC.     

Reversible regulation by magnesium of chick embryo fibroblast proliferation

The rate of 3H-thymidine incorporation and of cell proliferation in chick embryo fibroblast cultures are reduced coordinately when the [Mg2+] of the external medium is reduced below the physiological concentration of about 0.8 mM. These effects of moderately reduced [Mg2+] and the accompanying change in appearance of the cells, resemble the effects produced by lowering the [serum] of the medium. Cells subjected to severe Mg2+ deprivation, especially at low [Ca2+], die and detach from the culture dish. Cells kept at a reduced rate of proliferation for three days by moderate Mg2+ deprivation are quickly restored to rapid proliferation upon restoration of the normal [Mg2+] of the medium. The rate of proliferation of the chick embryo cells is reduced markedly by lowering [Ca2+] about 100-fold, but unlike the case of Mg2+-deprivation this can occur without significant effect on the rate of 3H-thymidine incorporation. More severe Ca2+ deprivation, which does lower the rate of 3H-thymidine incorporation, produces retraction of cells from one another and from the dish, and results in a distinctly abnormal, rounded appearance. The results lend weight to the thesis that free [Mg2+] plays a central role within the cell in the coordinate control of metabolism and growth. They also suggest that the effects produced by varying [Ca2+] in the medium are caused by changes at the external surface of the cell.     

Gadolinium promoted proliferation in mouse embryo fibroblast NIH3T3 cells through Rac and PI3K/Akt signaling pathways

Nephrogenic systemic fibrosis (NSF) is a fibrosing disorder disease developed in patients with underlying renal insufficiency following exposure to gadolinium-based contrast agents (GBCAs). Previous studies have demonstrated that GdCl₃ can promote NIH3T3 fibroblast cell proliferation, which provide a new clue to the role of GBCAs in the development of NSF. In the present study, we further clarify the molecular mechanism of Gd-promoted proliferation. The results showed that intervention with the Rac inhibitor NSC23766 abrogated Gd-promoted proliferation. The levels of active Rac1 significantly increased in Gd-treated cells detected by pull-down assays. In addition, the phosphorylation of Akt was significantly elevated in the treatment group, which was blocked by NSC23766. NSC23766 also reduced the migration of NIH3T3 cells enhanced by Gd. Moreover, the F-actin cytoskeleton was strengthened and the mitotic cell numbers was significantly increased after exposure to Gd. These results suggest that Rac and PI3K/Akt signaling pathways, as well as integrin-mediated signal pathway may play important roles in Gd-induced cell proliferation. In addition, under serum-free condition, Gd could decrease ROS accumulation and increase NIH3T3 cell survival.     

Rap2b promotes proliferation,migration, and invasion of lung cancer cells

Rap2b, a member of the guanosine triphosphate-binding proteins, is widely up-regulated in many types of tumors. However, the functional role of Rap2b in tumorigenesis of lung cancer remains to be fully elucidated. In this study, we investigated the effect of Rap2b on the lung cancer malignant phenotype, such as cell proliferation and metastasis. We found that Rap2b could promote the abilities of lung cancer cell wound healing, migration, and invasion via increasing matrix metalloproteinase-2 enzyme activity. Furthermore, Rap2b overexpression could increase the phosphorylation level of extracellular signal-regulated protein kinases 1/2. In conclusion, our results suggested that Rap2b may be a potential therapeutic target for lung cancer.     

Studies on serum stimulation of mouse fibroblast migration

Evidence is presented that the factor present in rat serum that promotes the migration of mouse fibroblasts (3T3) is a high mol. wt molecule. Amino acids, Mg²⁺ and Ca²⁺ are essential for full expression of activity. Once exposed to serum there is an 8–10 h lag period before cell migration can be detected. Cycloheximide, velban, colchicine, actinomycin D, and cytochalasin B, all inhibit cellular migration from a wound edge.     

Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells

Background  

We have demonstrated that in some human cancer cells both chronic mild heat and ionizing radiation exposures induce a transient block in S and G2 phases of the cell cycle. During this delay, cyclin B1 protein accumulates to supranormal levels, cyclin B1-dependent kinase is activated, and abrogation of the G2/M checkpoint control occurs resulting in mitotic catastrophe (MC).     

Overexpression of Hsc70 promotes proliferation,migration, and invasion of human glioma cells

Migration and invasion are often recognized as the main reasons for the high recurrence and death rates of glioma and limit the efficacy of surgery and other antitumor therapies. In this study, we found over activation of heat shock cognate protein 70 (Hsc70) in human glioma specimens, which was closely related to glioma grade. We investigated whether Hsc70 induced the migration and invasion of glioma cells. Wound healing and transwell migration assay were used to determine the migration and invasion ability of human glioma U251 and U87 cells, in which the expression of Hsc70 was knocked down by small interfering RNA. Western blot analysis was performed to determine the expression of FAK-Src signaling in malignant glioma cells. The results showed that Hsc70 deficiency significantly retarded migration and invasion and reduced the phosphorylation of FAK, Src, and Pyk2 in U251 and U87 cells. Overall, our results indicate that the migration and invasion capacity of human brain glioma cells is at least partly induced by Hsc70-dependent activation of FAK-Src signaling.     

Overexpression of human osteopontin increases cell proliferation and migration in human embryo kidney-293 cells

Malignant tumors are characterized by dysregulated cell growth and the metastasis of secondary tumors. Numerous studies have documented that osteopontin (OPN) plays a key role in regulating tumor progression and metastasis. Here, we show that the overexpression of OPN in human embryo kidney-293 cells significantly increases both the level of cell proliferation, by provoking the G₁/S transition, and the level of cell migration in vitro. These findings suggest that augmented OPN contributes to cell growth and motility. Inhibiting OPN or the pathway it stimulates may therefore represent a novel approach for the treatment of primary tumors and associated metastases.     

Glucocorticoid and thyroid hormones inhibit proliferation of serum-free mouse embryo (SFME) cells

Mouse embryo cells derived in a serum-free medium formulation (SFME cells) do not exhibit growth crisis or chromosomal abnormalities and are nontumorigenic in vivo; these cells are also reversibly growth inhibited by serum or platelet-free plasma (Loo et al.; Science, 236:200-202, 1987). A portion of the inhibitory activity of serum could be extracted by charcoal, a procedure that removes steroid and thyroid hormones. Both L-3,5,3'-triiodothyronine (T3) and hydrocortisone inhibited growth of SFME cells in a reversible manner. The inhibitory activity of serum also was partially removed by treatment with anion exchange resin in a procedure designed to deplete serum of thyroid hormone. However, the effect of serum on untransformed SFME cells could not be prevented by addition of the antiglucocorticoid RU38486, and ras-transformed clones of SFME cells, which are capable of growing in serum-containing medium, retained inhibitory responses to glucocorticoid and, with some clonal variability, to T3. These results suggest that glucocorticoid or thyroid hormones may contribute to the inhibitory activity of serum on SFME cells, but additional factors are also involved.     

Effect of tunicamycin on hexose transport in mouse embryo fibroblast Swiss 3T3 cells

The role of glycosylation of the carrier in the transporting activity was investigated in Swiss 3T3 cells. Inhibition of protein glycosylation by tunicamycin resulted in the decrease of hexose uptake in a dose- and time-dependent manner without a cytotoxic effect. From kinetic analysis, a decrease in the number or availability of hexose carriers in the plasma membrane was suggested. This was in good correlation with the decrease in the amount of photoaffinity cytochalasin B binding in the plasma membrane by the treatment with tunicamycin. The rate of phorbol 12,13-dibutyrate-induced translocation of the hexose carrier from microsomal to plasma membrane was reduced in tunicamycin-treated cells, which may be correlated with the decrease in the number of the completely glycosylated carrier translocatable from the microsomal membrane. In both tunicamycin-treated and untreated cells, the stimulation of hexose transport by phorbol 12,13-dibutyrate was abolished by the removal of phorbol 12,13-dibutyrate, and upon its readdition the stimulation recovered to the same degree as before the removal. Thus, the recycling of the functionally mature hexose carrier appeared not to be affected by the treatment with tunicamycin. These results suggested that complete glycosylation of the carrier may be necessary for the translocation of the carrier from microsomal to plasma membrane to accomplish its function on the cell surface.     

Effects of a low-intensity electromagnetic field on fibroblast migration and proliferation

The aim of this study was to test if an extremely weak 1?GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [(3)H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.     

Effects of a low-intensity electromagnetic field on fibroblast migration and proliferation

The aim of this study was to test if an extremely weak 1 GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [³H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.