Soluble Collagen VI Induces Tyrosine Phosphorylation of Paxillin and Focal Adhesion Kinase and Activates the MAP Kinase Erk2 in Fibroblasts

Signals from the extracellular matrix can modulate cellular differentiation and gene expression. We have shown previously that in contrast to other extracellular matrix molecules pepsin-solubilized collagen VI (CVI) can stimulate DNA synthesis of various mesenchymal cell types, apparently independent of integrin-mediated signal transduction. In order to further elucidate collagen VI-induced signaling events, we exposed mouse 3T3 fibroblasts and human HT1080 fibrosarcoma cells to soluble CVI. CVI induced tyrosine phosphorylation of proteins that associate with focal adhesions, such as paxillin, focal adhesion kinase (FAK), and p130CAS. Furthermore, it activated the mitogen-activated protein kinase, erk2. Kinetic analysis showed that these phosphorylations were transient, reaching a maximum after 5 min for transformed HT1080 cells and 30 min for 3T3 fibroblasts. These effects were partly inhibited by a beta1-integrin function blocking antibody and by single chains of CVI. Our results indicate that soluble fragments of native collagen VI, a ubiquitous component of the interstitial extracellular matrix, can mediate stimulation of DNA synthesis via tyrosine phosphorylation of paxillin, FAK, p130CAS, and erk2 in the absence of classical growth factors. Thus, CVI may serve as a matrix-derived sensor that allows for rapid reconstitution of a tissue defect by activating nearby mesenchymal cells.     

Collagen VI Regulates Normal and Transformed Mesenchymal Cell Proliferationin Vitro

Suggestions exist that, in addition to traditional growth factors, the extracellular matrix (ECM) of a cell can regulate its proliferation. This hypothesis was investigated with normal and transformed fibroblasts because they exhibit specific intracellular responses after adherence to ECM and produce large quantities of ECM proteins. Although cells cultured on different ECM proteins grew more rapidly than those on plastic, adherence and cell growth on an individual ECM protein were not correlated. To test if ECM can stimulate cell growth, soluble ECM proteins were given to cells after plating. In this culture system only collagen VI (CVI), at a concentration of 20 μg/ml in medium, increased 3T3 cell number to 402% of control by 72 h. Similar increases of human fibroblasts and HT 1080 cell numbers were noted. DNA synthesis of all three cell types increased 24 h after addition of soluble CVI. A mixture of CVI single chains, yielded by reduction and alkylation, was not stimulatory. However, this mixture efficiently inhibited the DNA synthesis induced by native CVI. Antibody inhibition studies showed that the region of CVI stimulating proliferation differs from the site bound by the integrin receptor α2β1, which mediates cell adhesion to immobilized CVI. Heparin inhibited a portion of CVI-induced proliferation. These data demonstrate that CVI can stimulate mesenchymal cell growth via a pathway that is independent of the integrin α2β1 and that the stimulatory region appears to be within the native helical portion of the collagen.     

Cell cycle analysis of in vitro cultured goral (Naemorhedus caudatus) adult skin fibroblasts

The present study was undertaken to examine cell cycle characteristics of endangered Goral (CITES Appendix I) adult skin fibroblasts. Seven experiments were performed, each with a one-way completely randomized design involving three replicates. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved, and fully confluent stages of Goral cells. In Experiments II and III, the effects of different antioxidants like beta-mercaptoethanol (beta-ME, 10 microM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 24 h and 4 h, respectively. In Experiments IV and V, three protease inhibitors, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 microg/ml) and cytochalasin B (7.5 microg/ml), were used as in Experiment II. In Experiments VI and VII, the effect of different levels of dimethylsulphoxide (DMSO) at 0%, 0.5%, 1.0% and 2.5% were tested by flow cytometry (FACS). In Experiment I, 68.7% of Goral skin fibroblasts reached the G(0)/G(1) stage (2C DNA content) when subjected to the serum-starved medium, which was more than the cycling (64.9%) and fully confluent groups (61.0%) (P > 0.05). Among the chemically treated group, beta-ME, cysteine and DMSO showed better results for synchronization of G(0) + G(1) phases than cycling, serum-starved and fully confluent groups. It can thus be concluded that beta-ME, cysteine and DMSO at certain concentrations can synchronize the cell cycle effectively, which could have a positive impact on somatic cell nuclear transfer in the goral.     

Regulation of collagen VI expression in fibroblasts. Effects of cell density, cell-matrix interactions, and chemical transformation

Collagen VI expression was studied in cultured human skin fibroblasts and mouse 3T3 cells using cDNA probes specific for alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. A 2-3-fold increase of these mRNAs was observed when fibroblasts were grown at increasing densities while only minimal changes occurred for the mRNA levels of collagens I and III, fibronectin, and beta-actin. Changes in mRNA correlated well with an increased production of corresponding proteins as determined by immunological assays. A comparable increase of alpha 1(VI) and alpha 2(VI) but not of alpha 3(VI) chain mRNAs was found for fibroblasts grown in a three-dimensional collagen gel after gel contraction. These conditions resulted, however, in a decrease of steady-state levels of collagens I and III and actin mRNAs. Transformation of 3T3 cells by phorbol ester did not change collagen VI mRNAs but caused a 3-5-fold reduction in mRNA levels for the other extracellular matrix proteins. These data strongly imply different regulatory mechanisms for the expression of collagen VI compared with collagens I and III and fibronectin. The differences may be correlated to changes in cell shape and reflect the requirement for collagen VI as a cell-binding protein.     

Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its alpha 2(VI) and alpha 3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded alpha 2(VI) and alpha 3(VI) chains but not on the alpha 1(VI) chain. The interactions with the chains were inhibited by low concentrations (10-100 microM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data indicate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.     

Induction of (2'-5')oligoadenylate synthetase in serum-starved HeLa S3 cells by growth factors and its role in growth regulation

When serum-starved HeLa S3 cells were stimulated to proliferate by addition of fetal calf serum (FCS), (2'-5')oligoadenylate synthetase (2-5A synthetase) activity was induced. Although no interferon (IFN) activity was detectable in the HeLa S3 cell-conditioned culture medium after growth stimulation, addition of anti-IFN-beta monoclonal antibody inhibited both the expression of the 2-5A synthetase gene and the production of the enzyme, suggesting that endogenous IFN-beta was involved in 2-5A synthetase induction. Purified preparations of three growth factors, epidermal growth factor, platelet-derived growth factor, and insulin, also induced 2-5A synthetase through IFN-beta. When serum-starved HeLa S3 cells were treated with FCS, DNA synthesis was initiated synchronously, with peaks after 12 and 32 h, although the level of 2-5A synthetase reached a maximum after the first peak of DNA synthesis. Inhibition of 2-5A synthetase induction by anti-IFN-beta antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa S3 cells, after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.     

Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its α2(VI) and α3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded α2(VI) and α3(VI) chains but not on the α1(VI) chain. The interactions with the chains were inhibited by low concentrations (10–100 μM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data incidate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.     

Expression of laminin by human fibroblasts, HT1080 fibrosarcoma cells and MCF-7 breast adenocarcinoma cells. Lack of regulation by the cell density and extracellular matrix.

We have cultured normal fibroblasts, fibrosarcoma HT1080 cells and breast adenocarcinoma MCF-7 cells on various substrates (plastic, collagen type I, laminin). All cell types used adhered on the three substrates with, however, a delayed attachment on laminin. On all substrates, cell grew as monolayer with the exception of MCF-7 cells that formed clusters on laminin. The epithelial MCF-7 cells as well as mesenchymal cells (fibroblasts and tumoral HT1080 cells) synthesized laminin and expressed mRNA coding for laminin B1 chain and for the 67 kD laminin binding protein. The levels of these mRNAs were not modulated by culture conditions which affect cell morphology nor by cell density.     

Bi-functional action of transforming growth factor-beta on DNA synthesis in early passage human fetal fibroblasts

We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.     

Stimulation of ornithine decarboxylase activity in chick fibroblasts by non-suppressible insulin-like activity (NSILA), insulin and serum.

A factor isolated from human serum (nonsuppressible insulin-like activity, NSILA) stimulates multiplication of serum-starved chick embryo fibroblasts and stimulates activity of ornithine decarboxylase (ODC). Physiological doses of NSILA (200 muU/ml) and pharmacological doses of insulin (200 mU/ml) stimulate ODC 4-5-fold, 10% fetal calf serum about 18-fold. Combined addition of NSILA and insulin does not result in higher activities, suggesting a common mechanism of action. The increase in cell number obtained with NSILA, insulin or serum parallels the degree of ODC stimulation. Treatment of cells with pronase also stimulates ODC activity. A sharp increase in ODC activity occurs between 2, 5 and 5.0 hours after addition of the growth factors with a peak at 4.0-4.5 hours ("activation period"). As cells leave G1 phase, ODC activity decreases rapidly. To achieve maximal activity of ODC, the growth factors have to be present during the entire "activation period." The potential to reactivate ODC decreases as cells pass through S phase. Results obtained using cycloheximide suggest that ODC is translated only in the second half of the "activation period." Data on effects of dbcAMP and dbcGMP on ODC activation by serum are discussed.     

Influence of serum on adult and fetal dermal fibroblast migration adhesion, and collagen expression

Summary The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater faction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.     

Contraction of fibroblast-containing collagen gels: Initial collagen concentration regulates the degree of contraction and cell survival

Remodeling of extracellular matrix involves a number of steps including the recruitment, accumulation, and eventual apoptosis of parenchymal cells as well as the production, organization, and rearrangement of extracellular matrix produced by these cells. The culture of fibroblasts in three-dimensional gels made of type I collagen has been used as a model of tissue contraction which characterizes both wound repair and fibrosis. The current study was designed to determine the effect of initial collagen concentration on the ability of fibroblasts to contract collagen gels and on cell survival. Native type I collagen was extracted from rat tail tendons and used to prepare collagen gels with varying collagen concentrations (0.75-2.0 mg/ml). Human lung fibroblasts (HFL-1) were cast into the gels and cultured in Dulbecco modified Eagle medium with 0.1% fetal calf serum for 2 wk. The gel size, collagen content, and deoxyribonucleic acid (DNA) content were determined. Gels prepared with an initial concentration of 0.75 mg/ml contracted more rapidly and to a smaller final size than gels prepared from 2 mg/ml initial collagen concentration (final size 7.1 versus 36.4% of initial size, P < 0.01). There was no significant degradation of the collagen in the gels under either condition. Hence, the dramatically increased contraction of the lower density gels resulted in a higher final density (P < 0.01). Cell density was estimated from DNA content. In low initial density gels, the final DNA content was significantly less than that in higher initial density gels (0.73 versus 1.88 microg/gel, P < 0.05). This was accompanied by an increased percentage of apoptotic cells at day 14 (43.3 versus 34.1%, P < 0.05). If the gels were maintained in the attached state which largely prevents contraction, apoptosis was significantly reduced, suggesting that contraction rather than matrix composition was a requirement for the increased apoptosis. In summary, these findings indicate that the initial matrix composition can lead to differing outcomes during fibroblast-mediated wound contraction.     

Developmental Block and Programmed Cell Death in Bos indicus Embryos: Effects of Protein Supplementation Source and Developmental Kinetics

The aims of this study were to determine if the protein source of the medium influences zebu embryo development and if developmental kinetics, developmental block and programmed cell death are related. The culture medium was supplemented with either fetal calf serum or bovine serum albumin. The embryos were classified as Fast (n = 1,235) or Slow (n = 485) based on the time required to reach the fourth cell cycle (48 h and 90 h post insemination - hpi -, respectively). The Slow group was further separated into two groups: those presenting exactly 4 cells at 48 hpi (Slow/4 cells) and those that reached the fourth cell cycle at 90 hpi (Slow). Blastocyst quality, DNA fragmentation, mitochondrial membrane potential and signs of apoptosis or necrosis were evaluated. The Slow group had higher incidence of developmental block than the Fast group. The embryos supplemented with fetal calf serum had lower quality. DNA fragmentation and mitochondrial membrane potential were absent in embryos at 48 hpi but present at 90 hpi. Early signs of apoptosis were more frequent in the Slow and Slow/4 cell groups than in the Fast group. We concluded that fetal calf serum reduces blastocyst development and quality, but the mechanism appears to be independent of DNA fragmentation. The apoptotic cells detected at 48 hpi reveal a possible mechanism of programmed cell death activation prior to genome activation. The apoptotic cells observed in the slow-developing embryos suggested a relationship between programmed cell death and embryonic developmental kinetics in zebu in vitro-produced embryos.     

Influence of adeno-associated virus on adherence and growth properties of normal cells.

It has been shown previously that infection of newly established cell cultures from malignant human tumors with adeno-associated parvovirus type 2 or type 5 results in growth arrest and cell death. Here we report that the additionally observed antiproliferative effect on diploid human fibroblasts is transient and is connected to a reduced number of cells in S phase. Progression through the cell cycle is disturbed either in G0/G1 or at the G1/S boundary, but an additional arrest in G2 cannot be excluded. DNA synthesis and cell proliferation are resumed when cells are recultured after loosening of cell-matrix adhesions by trypsin treatment. In contrast, they are not resumed by solely providing growth factors via higher amounts of fetal calf serum. The results suggest that cell adherence is altered in adeno-associated parvovirus-infected human embryo fibroblasts.     

Non-apoptotic signaling pathways activated by soluble Fas ligand in serum-starved human fibroblasts. Mitogen-activated protein kinases and NF-kappaB-dependent gene expression

Many Fas-expressing cells do not undergo cell death upon Fas stimulation. In the normal human diploid cell line GM6112, the addition of soluble Fas ligand (sFasL) leads to morphological signs of cell death in less than 1% of cells. Treatment of serum-starved GM6112 fibroblasts with sFasL resulted in a rapid and transient phosphorylation of ERK1/2 without a significant increase in JNK and p38 activities. Unless co-treated with the protein synthesis inhibitor anisomycin, sFasL did not show gene-inducing activity in cells maintained in complete medium. However, when cells were serum-starved for 4 days, treatment with sFasL alone induced interleukin-6 gene expression and, less strongly, interleukin-8 gene expression. Sensitization of the gene-inducing activity by serum starvation correlated with NF-kappaB activation by sFasL. Furthermore, we found that the expression of FADD and caspase-8 was significantly reduced in serum-starved cells, whereas the level of cFLIP remained unchanged. Transfection of GM6112 cells with the antisense caspase-8 expression construct sensitized cells toward sFasL-induced NF-kappaB-dependent reporter activation. Our results support the notion that a change in the ratio of cFLIP and caspase-8 may be responsible for turning on the Fas-activated NF-kappaB pathway, which otherwise is supplanted by the death-inducing pathway.     

Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells

Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 +/- 6.0 cells; serum-starved group: 86.8 +/- 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.     

Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line.

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.