Fetal calf ligament fibroblasts in culture secrete a low molecular weight collagen with a unique resistance to proteolytic degradation

A highly unusual collagen was secreted by fibroblasts cultured from 150- and 270-d-old fetal calf nuchal ligaments. Purification revealed that this protein (which may be synthesized in a higher molecular weight form) was precipitated at unusually high concentrations of ammonium sulfate and was also eluted from DEAE-cellulose at greater salt concentrations than were types I and III procollagens. On SDS PAGE, the collagenous protein exhibited an Mr of approximately 12,750 that was not altered in the presence of reducing agent. The low molecular weight collagen (FCL-1) was sensitive to bacterial collagenase and had a [3H]glycine content comparable to that found in type I procollagen, although the [3H]Hyp to [3H]Pro ratio was 0.43. FCL-1 was not cleaved by human skin collagenase, mast cell protease, trypsin, Staphylococcal V8 protease, or proteinase K at 37 degrees C. The collagen was susceptible to trypsin, but not to V8 protease, only after heating at 80 degrees C for 30 min. Preliminary structural studies indicate that FCL-1 was resistant to cleavage by CNBr but exhibited limited proteolysis with pepsin. Both 150- and 270-d-old fibroblasts produced comparable levels of interstitial (types I and III) procollagens, which comprised approximately 70% of the total protein secreted into the culture medium. However, 270-d-old (term) fibroblasts secreted approximately 50% more FCL-1, as percent of total culture medium protein, in comparison to the cells from the earlier gestational stage. This collagen may therefore play a role in the development of the nuchal ligament.     

Fibronectin-independent attachment of human gingival fibroblasts to interstitial and basement membrane collagens

We have studied the ability of human gingival fibroblasts (HGF) to attach to different interstitial (types I, II and III) and basement membrane (types IV and V) collagens. HGF cells were plated onto collagen-coated Petri dishes under various conditions and the percentage of cells attaching to the collagen was determined. HGF were found to attach to all the different types of native collagens, but attached poorly to the corresponding denatured collagens. When plated in the presence of 15% fetal bovine serum (FBS) or fibronectin-depleted FBS, similar percentages (approximately 85%) of cells attached to both interstitial and basement membrane collagens, demonstrating an attachment mechanism that is independent of plasma fibronectin. That the attachment in the presence of serum was also independent of cellular fibronectin was shown by the inability of fibronectin antibodies to block attachment to any of the collagen types. HGF were also capable of attaching to all of the collagen types in the complete absence of serum. In previous studies, investigators using cell lines have suggested that cell attachment in the absence of serum is non-physiological. However, the serum-free attachment of HGF to collagen was found to be dependent on cellular protein synthesis indicating that this attachment mechanism has biological significance.     

Soluble collagen VI drives serum-starved fibroblasts through S phase and prevents apoptosis via down-regulation of Bax

We previously showed that soluble, pepsin-solubilized collagen VI increases de novo DNA synthesis in serum-starved HT1080 and 3T3 fibroblasts up to 100-fold compared with soluble collagen I, reaching 80% of the stimulation caused by 10% fetal calf serum. Here we show that collagen VI also inhibits apoptotic cell death in serum-starved cells as evidenced by morphological criteria, DNA laddering, complementary apoptosis assays (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorting), and quantification of apoptosis-regulating proteins. In the presence of starving medium alone or collagen I, the proapoptotic Bax was up-regulated 2-2.5-fold, compared with soluble collagen VI and fetal calf serum, whereas levels of the antiapoptotic Bcl-2 protein remained unaffected. In accordance with its potent stimulation of DNA synthesis, soluble collagen VI carries serum-starved HT1080 and Balb 3T3 fibroblasts through G(2) as shown by fluorescence-activated cell sorting analysis, whereas cells exposed to medium and collagen I where arrested at G(1)-S. This was accompanied by a 2-3-fold increase in cyclin A, B, and D1 protein expression. Collagen VI-induced inhibition of apoptotic cell death may be operative during embryogenesis, wound healing, and fibrosis when elevated tissue and blood levels of collagen VI are observed, thus initiating a feedback loop of mesenchymal cell activation and proliferation.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Comparison of colony-formation efficiency of bovine fetal fibroblast cell lines cultured with low oxygen, hydrocortisone, L-carnosine, bFGF, or different levels of FBS

A comparison of colony-formation efficiency (CFE) was made between six independent bovine fetal fibroblast (BFF) cell lines used in somatic cell nuclear transfer. Variation in CFE was assessed under different culture conditions. The conditions examined were ambient atmosphere (approximately 20% oxygen) culture versus 5% oxygen culture, three levels of fetal bovine serum (FBS) in the medium (5%, 10% or 20%), and the amendment of 10% FBS medium with basic fibroblast growth factor (1 ng/mL), L-carnosine (20 mM), or hydrocortisone (1 microM). The six BFF cell lines showed significant differences from one another in CFE. No significant difference in CFE was found with reduced oxygen culture. L-Carnosine also had no significant effect on CFE. A FBS concentration of 10% was found to produce the best overall CFE. Hydrocortisone treatment reduced the size of colonies although the number of colonies formed was not affected. Basic FGF increased the size of colonies but the number of colonies formed was not affected. The results showed that different BFF cell lines varied significantly in their CFE. Also, some medium supplements or culture conditions that have shown positive CFE effects on the fibroblasts of other species failed to show significant positive CFE effects on the BFF cell lines tested.     

Effect of acute serum depletion on Na+-K+ homeostasis in cultured human skin fibroblasts

In order to elucidate changes in cell transport behavior of cultured human skin fibroblasts in response to acute serum depletion, we performed uptake and washout of 22Na+ and 86Rb+ as well as measurements of the intracellular Na+ and K+ levels in the presence and absence of ouabain. Pronounced and lasting increase in cellular Na+ and decrease in K+ were observed after removal of fetal bovine serum (FBS) from the medium. The sum of the Na+ and K+ contents (nEq/10(5) cells) was lower in FBS-free medium (mean +/- SD; 17.3 +/- 2.2) than in FBS-containing medium (26.2 +/- 3.8; P less than .02). Simultaneously, a decrease in cellular water volume was detected in the FBS-free medium. The cation uptake and washout data suggest that FBS removal primarily renders the cells more permeable to Na+ and K+ with a secondary stimulation of the ouabain-sensitive Na+ extrusion mechanism. FBS at a concentration of 0.2% prevented approximately 50% of the maximal increase in the 86Rb+ washout rate constant associated with FBS depletion. Ouabain (2 microM) produced an increase in the 86Rb+ washout rate constant. This effect was substantially larger in cells subjected to medium without FBS (from 0.0303 to 0.2500 min-1) than in fibroblasts incubated in medium with FBS (from 0.0107 to 0.0487 min-1). The cellular K+ content was drastically reduced by ouabain to a level not different in medium with or without FBS (33.9 +/- 4.5 to 1.75 +/- 0.38 and 16.7 +/- 1.4 to 1.4 +/- 0.13 nEq/10(5) cells, respectively). The 22Na+ washout data exhibited a three-exponential pattern. Analytical solutions of the washout data by means of two models (serial and parallel) with three compartments showed that FBS depletion resulted in increase of the size of all three compartments. It is concluded that in cultured human skin fibroblasts, FBS is essential to the maintenance of a normal Na+ and K+ homeostasis. The removal of FBS results in dramatic permutation of this homeostasis that develops within minutes and lasts for hours.     

Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin

Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.     

Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of α-smooth muscle (α-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, α-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor β (TGFβ) in FBS and PCS was determined and the effect of neutralizing anti-TGFβ antibody was evaluated. It was determined that FBS contained more TGFβ than PCS, but that essentially all the TGFβ was latent in both sera. However, results obtained with anti-TGFβ antibody show that active TGFβ is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFβ or an enzyme that is able to activate latent serum TGFβ. Alternatively, sera may contain two different forms of latent TGFβ, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds. J. Cell. Physiol. 171:1–10, 1997. © 1997 Wiley-Liss, Inc.     

Significance of α-SMA in myofibroblasts emerging in renal tubulointerstitial fibrosis

Myofibroblast transdifferentiation plays a crucial role in the development and progression of renal tubulointerstitial fibrosis. However, the significance of α-smooth muscle actin (α-SMA) expression, which is the major morphological characteristic of myofibroblasts, remains to be determined in detail. The effect of α-SMA expression on fibrosis tissue was examined by using a fibrosis model (collagen gel) in vitro. The transdifferentiation of fibroblasts into myofibroblasts was triggered in the culture medium with 0.5% fetal bovine serum (FBS)+transforming growth factor (TGF)-β1, but not with 10% FBS+TGF-β1. The TGF-β1-induced gel contraction caused by myofibroblasts was greater than that by fibroblasts. Gel contraction by myofibroblasts involved the Ca2+-dependent myosin light chain kinase pathway, as well as the activation of Rho kinase and p38 mitogen-activated protein kinase (MAPK). Taken together, these findings suggest that α-SMA expression in renal interstitial fibroblasts, i.e., myofibroblast transdifferentiation, accelerates the contraction of the tubulointerstitial fibrosis tissue via the Ca2+-dependent pathway, in addition to the pathways involved in fibroblast contraction; this event may lead to renal atrophy and renal failure.     

AcSDKP对PDGF诱导的大鼠心脏成纤维细胞增殖和胶原合成的调节作用

目的：探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酰（AcSDKP）对血小板源性生长因子（PDGF）诱导的大鼠心脏成纤维细胞增殖和胶原合成的调节作用。方法：建立新生大鼠心脏成纤维细胞系;采用四甲基偶氮唑（MTT）法和^3H-TdR掺入法检测心脏成纤维细胞的增殖;采用^3H-脯氨酸掺入法检测心脏成纤维细胞胶原的合成。结果：PD3F在1～20ng／ml浓度范围内对心脏成纤维细胞增殖和胶原合成均有促进作用。且随着PDGF浓度的增加,其促细胞增殖和胶原合成作用增强,并在10ng／ml浓度时PDGF的促增殖和胶原合成效应最强。在10^-10～10^-8mol／L浓度范围内,AcSDKP对PDGF介导的心脏成纤维细胞增殖和胶原合成均有抑制作用,并且在10叫mol／L时,AcSDKP抑制心脏成纤维细胞增殖和胶原合成作用最强。结论：AcSDKP对PDGF介导的心脏成纤维增殖和胶原合成均有明显抑制作用,这可能与其抗心脏纤维化的作用相关。     

Temperature dependence of collagen fluorescence.

Dermal collagens have several fluorescent moieties in the UV and visible spectral regions that may serve as molecular probes of collagen. We studied the temperature-dependence of a commercial calf skin collagen and acid-extracted Skh-1 hairless mouse collagen at temperatures from 9 degrees C to 60 degrees C for excitation/emission wavelengths 270/305 nm (tyrosine), 270/360 nm (excimer-like aggregated species), 325/400 nm (dityrosine) and 370/450 nm (glycation adduct). L-tyrosine (1 x 10(-5) M in 0.5 M HOAc) acted as a "reference compound" devoid of any collagen structural effects. In general, the fluorescence efficiency of these fluorophores decreases with increasing temperature. Assuming that rate constant for fluorescence deactivation has the form k(d)(T) = k(d) degrees exp (-DeltaE/RT), an Arrhenius plot of log[(1/Phi) - 1] vs. 1/T affords a straight line whose (negative) slope is proportional to the activation energy, DeltaE, of the radiationless process(es) that compete with fluorescence. Because it is difficult to accurately measure Phi(f) for collagen-bound fluorophores, we derived an approximate formula for an activation parameter, DeltaE*, evaluated from an Arrhenius-like plot of log 1/I(N)vs. 1/T, (1/I(N)vs. is the reciprocal normalized fluorescence intensity). Tyrosine in dilute solution affords a linear Arrhenius plot in both of the above cases. Using the known value of Phi(f) = 0.21 for free tyrosine at room temperature, we determined that DeltaE* is accurate to approximately 25% in the present instance. Collagen curves are non-linear, but they are quasi-linear below approximately 20 degrees C, where the helical form predominates. Values of DeltaE* determined from the data at T < 20 degrees C ranged from 6.2-8.4 kJ mol(-1) (1.5-2.0 kcal mol(-1)) for mouse collagen and 10.3-11.4 kJ mol(-1) (2.5-2.7 kcal mol(-1)) for calf skin collagen, consistent with collisional deactivation of the fluorescent state via thermally enhanced molecular vibrations and rotations. Above 20 degrees C, log 1/I(N)vs. 1/T plots from Skh-1 hairless mouse collagen are concave-downward, suggesting that fluorescence deactivation from the denatured coil has a significant temperature-independent component. For calf skin collagen, these plots are concave-upward, suggesting an increase in activation energy above Tm. These results suggest that collagen backbone and supramolecular structure can influence the temperature dependence of the bound fluorophores, indicating the future possibility of using activation data as a probe of supramolecular structure and conformation.     

Influence of increased extracellular calcium concentration and donor age on density-dependent growth inhibition of human fibroblasts

Elevated calcium chloride concentration [( CaCl2]) has been shown to increase saturation density for an established mouse fibroblast line and for human fetal lung fibroblasts (WI-38). In order to examine the effect of increased [CaCl2] on human fibroblasts from donors of varying age, fibroblasts were grown in medium (basal level of 1.8 mM CaCl2) supplemented with fetal bovine serum (FBS) until confluent. Compared to controls in basal medium, newborn foreskin fibroblasts exposed to additional CaCl2 had a 110-450% increased cell yield that was independent of [CaCl2] within the range of an additional 1.5-5.0 mM. The effect was maintained over an eightfold range of FBS concentration. Initial growth rate was unaffected, but a prolongation of exponential phase occurred for cultures exposed to increased [CaCl2]. Confluent cultures refed medium with increased [CaCl2] were stimulated 5- to 10-fold more than cultures refed basal medium. An additional 2 mM CaCl2 resulted in a 210% increase for young adult-derived fibroblasts versus a 29% increase for old adult-derived fibroblasts (P less than 0.001). These data indicate that increased [CaCl2] decreases density-dependent growth inhibition of postnatal human dermal fibroblasts in vitro and that this effect is donor age dependent.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured fibroblasts from patients with mevalonate kinase deficiency: differential response to lipid supplied by fetal bovine serum in tissue culture medium

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was measured in extracts of cultured fibroblasts derived from patients with mevalonate kinase deficiency (MKD). For six patients studied, the mean activity of 63.3 +/- 41.1 pmol/min-mg protein (+/- 1 SD, range 37.7-146.2) was significantly higher than the mean value in three control fibroblast lines of 11.1 +/- 3.5 (+/- 1 SD, range 8.0-14.9). These values were obtained using cells subcultured in medium supplemented with 10% fetal bovine serum (FBS) 21 h prior to assay. When cells were deprived of cholesterol by subculturing for 21 h in delipidated FBS, the mean value for patient cells was increased to 230.8 +/- 78.5 pmol/min-mg protein (range 130.9-333.8) as compared to 109.5 +/- 47.1 (range 78.0-163.6) for controls. The activity of HMG-CoA synthase in extracts of fibroblasts derived from the patients was not elevated. The mevalonic acid concentration in the surrounding culture medium was assessed by stable isotope dilution assay. For five patients, the mean concentration in medium containing FBS was 0.92 +/- 0.37 microM (+/- 1 SD, range 0.46-1.48) in contrast to 1.24 +/- 0.83 microM (range 0.46-2.54) for cells subcultured in delipidated FBS. The mean value for three control fibroblast lines was 0.22 +/- 0.12 microM (+/- 1 SD, range 0.11-0.35) for cells subcultured in FBS as compared to 0.01 +/- 0.01 microM (range 0.0-0.01 microM) for cells sucultured in delipidated FBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of human serum on human fetal skin fibroblast migration: Migration-inhibitory activity and substances in serum, and its age-related changes

Summary In order to clarify the environmental factors modulating cell migration, we investigated the effects of human serum on cell migration, and found that serum from adult donors strongly (by 48%) suppressed the migration of human fetal skin fibroblasts into a denuded area in a cell monolayer. Human serum from old donors inhibited cell migration more strongly than that from adult donors. Next, we investigated the properties of migration-inhibitory activity of human serum and serum proteins in order to identify migration-inhibitory substances. Human serum from adult donors strongly suppressed the migration of human fetal skin fibroblasts, although it stimulated cell proliferation more strongly than fetal bovine serum (FBS), indicating that the inhibitory effects of human serum on cell migration was not due to its toxic effects. The inhibition of cell migration by human serum was concentration dependent. It was demonsstrated that the inhibition did not depend on the inhibitory effects of human serum on collagen synthesis. The migration-inhibitory activity was seen in fractions over 100 kDa, as determined by an ultrafiltration membrane, and no inhibitory activity was observed in fractions under 100 kDa. On the other hand, it was not detected either in fractions over 100 kDa or under 100 kDa in FBS. Among the over 100 kDa human serum proteins examined, γ-globulin, α2-macroglobulin, and low density lipoprotein (LDL) suppressed fibroblast migration in a concentration-dependent manner. However, among the three, cell migration-inhibiting activity of γ-globulin almost disappeared when cell migration was conducted in 10% FBS-supplemented medium. These results indicated that α2-macroglobulin and LDL were candidate substances for cell migration-inhibiting activity in human serum.     

Similar, but not identical, modulation of expression of extracellular matrix components during in vitro and in vivo aging of human skin fibroblasts.

Regulation of the synthesis of procollagen and other extracellular matrix components was examined in human skin fibroblasts obtained from donors of various ages, from fetal to 80 years old (in vivo aged), and in fetal fibroblasts at varying passage levels (in vitro aged). Growth rates and saturation densities of fibroblasts decreased with increasing age of the donor and after passage 20 of fetal fibroblasts. The rates of collagen and proteoglycan synthesis also decreased during both types of aging to about 10-25% of the rate in early passage fetal fibroblasts, whereas the synthesis of total noncollagenous proteins was not greatly affected. Decreased collagen synthesis in both types of aging was correlated with lower steady-state levels of mRNAs for the two subunits of type I procollagen mRNA, although their regulation was not coordinate. Type III collagen mRNA levels also declined in both types of aging. The concentration of fibronectin mRNA also decreased during in vitro aging but more rapidly than the collagen mRNAs, whereas in fibroblasts from 51-80-year-old donors, it was similar to or higher than in early passage fetal fibroblasts. This study suggests that the decreased synthesis of procollagen and proteoglycans in in vivo aged fibroblasts represents changes that are responsible for intrinsic degenerative changes that occur in human skin during aging. Furthermore, although in vitro and in vivo aging were similar in many respects, they were not equivalent, as evidenced by the differences in regulation of fibronectin expression.     

Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro

The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.     

Calcium, lanthanum, pyrophosphate, and hydroxyapatite: a comparative study in fibroblast mitogenicity

Calcium-containing crystals and elevated levels of calcium chloride (CaCl2) and lanthanum chloride (LaCl3) have been previously reported to enhance the proliferative activity of cultured fibroblasts. We have investigated the relative mitogenicity of these agents, whether they function via precipitation on the cell surface and whether they interact with one another. Confluent cultures of newborn foreskin fibroblasts provided with fresh medium containing 10% fetal bovine serum (FBS) in the presence of hydroxyapatite (HA), pyrophosphate (PPi), LaCl3 (La), or additional CaCl2 (Ca) were all stimulated more than control cultures provided with fresh medium and 10% FBS alone as assessed by cell counts 5 days later. Increases in cell yield above the original confluent cell density were 316% for La, 271% for Ca, 189% for HA, 131% for PPi, and 45% for controls. Addition of fresh medium containing 10% FBS and epidermal growth factor or fresh medium containing 20% FBS as additional points of reference yielded increases of 204 and 107%, respectively, over original confluent density. Stimulation induced by La or Ca was significantly greater (P less than 0.001) than the stimulation induced by each of the other treatments. The same treatments added to confluent cultures without a change of medium also renewed mitotic activity, with La and Ca again the most mitogenic and approximately doubling the pretreatment cell yields. Cultures incubated in an inverted position to avoid cell contact with precipitates in the medium were also stimulated by La and Ca, but not by HA and PPi. When added to confluent cultures simultaneously supplemented with optimal additional Ca, La decreased Day 5 cell yields in a dose-dependent manner at low concentrations (0.03-0.2 mM) but increased cell yields over those obtained with 0.2 mM LaCl3 again in a dose-dependent manner at higher concentrations. Thus, while HA and PPi act via precipitation on the cell surface, the more mitogenic agents La and Ca function in solution and appear to stimulate cell division by different nonadditive mechanisms. These findings suggest multiple mechanisms of membrane participation in mitogen responsiveness and in density-dependent inhibition of growth.     

Studies on the role and mode of operation of the very-lysine-rich histone H1 in eukaryote chromatin. The isolation of the globular and non-globular regions of the histone H1 molecule.

Digestion of calf thymus H1 histone with thrombin cleaves the molecule at the sequence -(Pro)-Lys-Lys-Ala-, corresponding to a point approximately 122 residues from the N-terminus (about 56% along the molecule). The N-terminal fragment is shown by proton nuclear magnetic resonance (NMR) to possess the globular structure of the intact histome H1 molecule, whereas the C-terminal fragment appears to possess little or no structure. The N-terminal fragment separates into two peaks on an ion-exchange column, one of which is shown to originate from a single subfraction of calf thymus histone H1 and the other to originate from the other subfractions, by detailed comparison of the NMR spectra. It thus seems that the structure of the H1 histone in solution under physiological conditions consists of a globular head with a highly basic random coil tail. It is suggested that the globular head has a specific binding site on the subunit structure of the chromosome.