Remodeling of actin cytoskeleton in mouse periosteal cells under mechanical loading induces periosteal cell proliferation during bone formation

Background

The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo.

Principal Findings

We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells.

Conclusions

These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation.     

Clenbuterol treatment stimulates cell proliferation in denervated chick gastrocnemius muscle

Daily administration of clenbuterol, a beta adrenoceptor agonist (0.5 mg/kg body weight; for 7 days) to normal innervated and denervated adult chicks (Gallus domesticus) resulted in different growth related responses by gastrocnemius muscle. While normal innervated muscle undergoes hypertrophy, structural reorganization in denervated tissue is accomplished by the de novo emergence of new cells. A contrasting cell population with extremely narrow cross sectional dimensions is a characteristic feature of denervated muscle in presence of clenbuterol. Measurement of fiber dimensions, number of cells per unit area and examination of histochemical preparations support this.     

Estrogen-mediated cell proliferation during chick oviduct development and its modulation by progesterone

The interactions of estrogen and progesterone on mitosis were examined in the surface epithelium of the developing chick oviduct. Both of these steroid hormones can stimulate cells to divide in the unstimulated oviduct. However, progesterone treatment results in a delayed suppression of cell division in both the presence and absence of estrogen. This progesterone induced depression of estrogen-mediated cell division is observed throughout oviduct development. During oviduct development estrogen is necessary for both cell division and the differentiation of specific cell types while progesterone appears to modify the action of estrogen by blocking the progression of cells through the cell cycle.     

Oxytocin stimulates proliferation of human osteoblast-like cells

Oxytocin receptors have recently been demonstrated in human osteoblast-like (hOB) cells. In this study, oxytocin 100-1000 pmol/l increased cell proliferation of primary cultures of hOB cells, measured by [3H]thymidine incorporation, (P<0.01). In human osteosarcoma cell-line (SaOS-2), oxytocin 100 pmol/l increased cell proliferation (measured by [3H]thymidine incorporation and a commercially available kit) and protein synthesis ([3H]proline incorporation) (P<0.05). The increase in cell proliferation was abolished when SaOS-2 cells were incubated with an oxytocin antagonist and oxytocin. Oxytocin 100 pmol/l decreased interleukin-6 (IL-6) production of the hOB cells (23.4+/-1.96 versus 33.4+/-2.65 pg/well; P<0.001). These findings indicate that oxytocin may affect bone metabolism in humans.     

DNA repair in lens cells during chick embryo development.

When chick lens epithelium is cultured in vitro, differentiation into lens fiber cells is accompanied by DNA degradation. This phenomenom of terminal differentiation was studied in the epithelium from embryos at the 6th and 11th days of development. DNA size and the ability of the cells to repair DNA damage induced by X-rays were analysed in alkaline sucrose gradients. In the 6-day epithelium a rapid degradation and complete lack of DNA repair were recorded. Similar observations have been made in previous studies on the 11-day sample, but here degradation is progressive and occurs after a lag of several days. In the younger epithelium, internal irradiation by [3H]thymidine also had a drastic effect resembling that caused by X-rays. In order to assess the process of differentiation in our experimental system the synthesis of delta- and alpha-crystallins was monitored. Stage-related modifications in the rates of synthesis were recorded. The results confirm that the DNA repair system is impaired during terminal differentiation. The differences observed between the two stages may reflect either a developmental modification in DNA repair mechanisms or a change in the relative proportions of differentiating cells. An hypothesis is proposed in support of the latter case.     

Activin stimulates proliferation of rat ovarian thecal-interstitial cells

There is growing evidence that the function of ovarian theca-interstitial (T-I) cells may be modulated by paracrine actions of activin, inhibin, and follistatin. Furthermore, either dysregulation, dysfunction, or both, of these peptides may play a role in conditions associated with T-I hyperplasia, such as polycystic ovary syndrome (PCOS) and hyperthecosis. This study was designed to evaluate the role of activin, inhibin, and follistatin in the modulation of T-I cell proliferation. Interaction of these peptides with insulin-like growth factor-I (IGF-I), a known stimulator of T-I cell proliferation, was also assessed. Purified rat T-I cells were cultured for 48 h in chemically defined media and with or without activin (3-30 ng/ml), inhibin (3-30 ng/ml), follistatin (100 ng/ml), and/or IGF-I (10 nM). T-I cell proliferation was assessed using radiolabeled thymidine incorporation assay. Activin alone stimulated proliferation of T-I cells in a dose-dependent fashion (by up to 320% above control; P < 0.001), whereas inhibin alone or follistatin alone had no significant effect. Inhibin had also no effect on activin-induced proliferation. Follistatin significantly reduced the stimulatory effects of activin and decreased proliferation by up to 46% (P < 0.01) below the level attained in the presence of activin alone. IGF-I (10 nM), at a dose producing a near-maximal effect, increased proliferation by 175% above control (P < 0.001); insulin (10 nM) increased proliferation by 52% above control (P < 0.03). A combination of IGF-I (10 nM) and activin (30 ng/ml) resulted in a 1090% increase of proliferation above control (P < 0.001); this stimulatory effect was significantly greater than that achieved in the presence of either activin alone or IGF-I alone (P < 0.001). Similarly, a combination of insulin (10 nM) and activin (30 ng/ml) increased proliferation by 506% above control levels. Flow cytometry evaluation revealed that activin increased the proportion of actively dividing cells (in S or G2/M phase of the cell cycle) by 42% (P < 0.02), whereas IGF-I had no effect on the proportion of actively dividing cells. The present findings indicate that an activin-follistatin system may be involved in the regulation of the size of ovarian thecal-stromal compartment. In view of the synergy between activin and IGF-I, and the difference in the effects on the cell cycle distribution, stimulation of T-I proliferation by these agents is likely to be mediated via separate transduction pathways. Excess activin or insufficient follistatin may contribute to T-I hyperplasia.     

Neuregulin stimulates DNA synthesis in embryonic chick heart cells.

Neuregulins are a family of growth factors that have been shown to promote the growth or differentiation of various cell types. Recently, targeted mutations of the genes for neuregulins or their putative receptors by homologous recombination resulted in embryonic lethality characterized by cardiac malformation. Here we investigate a role for neuregulin in the growth of cultured chick heart cells. Neuregulin induced the tyrosine phosphorylation of a 185-kDa protein in cultured heart cells, and it also stimulated an increase in [(3)H]thymidine incorporation and BrDU labeling in the cell cultures. Immunocytochemistry revealed that the increased DNA synthesis was primarily in mesenchymal cells and not detected in myocytes or endocardial cells. These data suggest that neuregulin may function as a paracrine signal in mesenchymal-endothelial interactions during cardiac development.     

The characteristics of the stimulation of the proliferation of cultured chick embryo cells during reseeding

The growth rate of chick embryo cells in slowly proliferating cultures is activated after substitution of conditioned medium by fresh one with serum. After cell replanting, the stimulation of cell proliferation takes place in both media with the same effectiveness. In serum-less medium replanted cells do not adhere to glass and die. The results suggest that cells reversibly lose their dependence upon serum growth factors and population density, as a result of replanting, but retain anchorage-dependence for growth.     

Mannose-6-phosphate stimulates proliferation of neuronal precursor cells

The mitogenic signal function of mannose-6-phosphate (Man-6-P)/insulin-like growth factor II (IGF-II) receptors was studied in neuronal precursor cells from developing rat brain (E15). About 30% of the cellular Man-6-P/IGF-II receptors were present on the cell surface. Man-6-P and IGF-II stimulated DNA synthesis twofold and their effects were additive. Antibody 3637 to the Man-6-P/IGF-II receptor blocked the response to Man-6-P but not that to IGF-II. Other phosphorylated hexoses were also active. Fructose-1-phosphate was equally potent with Man-6-P, whereas glucose-6-phosphate was 5 times less potent. We conclude that Man-6-P-containing proteins and IGF-II act as mitogens in developing brain by interaction with the Man-6-P/IGF-II receptor and the IGF-I receptor, respectively.     

Copper stimulates proliferation of human endothelial cells under culture

Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 μM CuSO₄ in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-α, transforming growth factor-β, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1α. Moreover, despite the previous observations that copper increased total specific binding of ¹²⁵I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis. J. Cell. Biochem. 69:326–335, 1998. © 1998 Wiley-Liss, Inc.     

beta-N-acetylhexosaminidase isoenzymes during chick embryo development

1. Two forms (I and II) with acidic pH optima and a neutral form of beta-hexosaminidase has been separated by DEAE-cellulose chromatography and characterized in skin and lung of 7, 9, 11, 14 day chick embryos and 1 day old chicken. 2. Forms I and II are similar to hexosaminidase A and B for their behaviour on DEAE-cellulose chromatography, Concanavalin A-Sepharose column and thermal stability. 3. Neutral form has a neutral pH optimum and higher molecular weight and a more acidic I. P. than forms I and II, a low beta-N-acetylgalactosaminidase activity and it is not bound by a Concanavalin A-Sepharose column and in that resemble hexosaminidase C and/or other neutral hexosaminidases. 4. We have found differences in the percentage of neutral form and in the specific activities of the extracts in the skin in different stages of development. 5. No significant differences were observed in the lung.     

Changes in the electrophoretic mobility of cells from chick blastoderms during early development

A change in cell surface charge density during early avian development is shown with a free-flow electrophoresis apparatus. The blastoderms of freshly laid eggs consist of two electrophoretically distinct cell populations. After the onset of gastrulation a third cell population with an intermediate electrophoretic mobility appears. With increasing time of incubation there is a shift in the proportion of these populations and an increase in the mobility of the fastest fraction.     

Culture supernatant of Lactobacillus acidophilus stimulates proliferation of embryonic cells

Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed chemotactic and angiogenic properties. Specifically, LS stimulated gene expression and the secretion of tumor necrosis factor-alpha (TNF-alpha), the proliferation of immune cells in vitro, and blood vessel formation. Chemotaxis and proliferation of inflammatory cells in vivo were also stimulated by LS. In the current study, we hypothesized that LS stimulates the growth and development of other rapidly dividing cells, including embryonic cells. The stimulatory effects of LS on a neuroblastoma cell line (Neuro-2a), chicken embryos, and bovine embryos were examined. The addition of LS to Neuro-2a cultures caused a proliferation of cells in a concentration-dependent manner. Pretreatment of LS at 56 degrees C for 30 mins did not affect its stimulatory activity. The administration of LS to the chorioallantoic membrane (CAM) of chicken-embryonated eggs for 1-2 days resulted in extensive thickening of the membrane. The thickening was due to the influx and proliferation of fibroblasts and inflammatory cells, the accumulation of loose connective tissue composed primarily of mucopolysaccharides, and/or the formation of blood vessels. Stimulatory effects of LS on bovine embryos were also observed. The treatment with LS significantly promoted the development of zygotes to the four-cell stage and from the four-cell stage to blastocysts. These results have confirmed our hypothesis that LS exerts a stimulatory effect on the cells of embryonic stages including neuroblastoma cells, the CAM of chicken embryos, and bovine embryos from zygotes to blastocysts.