Biomedical subjects

A Philip

Publications and source records attributed to A Philip.

At least 19 recordsLinked to original sources

PubMed2006

Expression and function of TbetaRII-B, a variant of the type II TGF-beta receptor, in human chondrocytes.

OBJECTIVE: Transforming growth factor-beta (TGF-beta) has profound effects on chondrocyte proliferation and matrix production, and dysregulation of TGF-beta action has been implicated in osteoarthritis. The mechanisms by which the diverse actions of TGF-beta are regulated in chondrocytes are unclear. Although it is well documented that TGF-beta signaling is transduced by types I and II receptors, other TGF-beta receptors may play critical roles by regulating signaling receptor activity. Our objective was to examine the expression of TbetaRII-B, a splice variant of the type II TGF-beta receptor, and to analyze its role in regulating TGF-beta signaling in human chondrocytes. METHODS: TbetaRII-B expression was examined in human cartilage tissue specimens, human chondrocyte cell lines C28/I2 and tsT/AC62, and human primary chondrocytes by Western blot and reverse-transcriptase-polymerase chain reaction. Ligand binding and heteromerization of TbetaRII-B with other TGF-beta receptors on the cell surface were analyzed by affinity labeling, immunoprecipitation, and two-dimensional SDS-PAGE. Regulation of TGF-beta responses by TbetaRII-B was determined by examining Smad2 phosphorylation, Smad3-specific signaling, transcriptional activity, and type II collagen levels. RESULTS: TbetaRII-B is expressed in normal and osteoarthritic human cartilage. Furthermore, it is a dynamic component of the TGF-beta receptor system in human chondrocytes, forming heteromeric complexes with the types I and II TGF-beta receptors, betaglycan and endoglin. Importantly, overexpression of TbetaRII-B leads to enhanced TGF-beta signaling and responses in chondrocytes. CONCLUSIONS: These results suggest that TbetaRII-B may play a key role in the regulation of TGF-beta action in human chondrocytes.

Cartilage, Articular↗

PubMed2001

Protein-tyrosine kinase Pyk2 mediates endothelin-induced p38 MAPK activation in glomerular mesangial cells.

Endothelin-1 (ET-1), a member of a family of 21 amino acid peptides possessing vasoconstrictor properties, is known to stimulate mesangial cell proliferation. In this study, ET-1 (100 nm) induced a rapid activation of p21(ras) in human glomerular mesangial cells (HMC). Inhibition of Src family tyrosine kinase activation with [4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or chelation of intracellular free calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester significantly decreased ET-1dependent p21(ras) activation and suggested the involvement of the cytoplasmic proline-rich tyrosine kinase Pyk2. We have observed that Pyk2 was expressed in HMC and was tyrosine-phosphorylated within 5 min of ET-1 treatment. ET-1-induced activation of Pyk2 was further confirmed using phospho-specific anti-Pyk2 antibodies. Surprisingly, Src kinase activity was required upstream of ET-1-induced autophosphorylation of Pyk2. To determine whether Pyk2 autophosphorylation mediated ET-1-dependent p21(ras) activation, adenovirus-mediated transfer was employed to express a dominant-negative form of Pyk2 (CRNK). CRNK expression inhibited ET-1-induced endogenous Pyk2 autophosphorylation, but did not abolish ET-1-mediated increases in GTP-bound p21(ras) levels. ET-1-induced activation of the p38 MAPK (but not ERK) pathway was inhibited in HMC and in rat glomerular mesangial cells expressing the dominant-negative form of Pyk2. These findings suggest that the engagement of Pyk2 is important for ET-1-mediated p38 MAPK activation and hence the biological effect of this peptide in mesangial cells.

Adenoviridae↗

PubMed2001

Fetal and adult human skin fibroblasts display intrinsic differences in contractile capacity.

One of the differences between fetal and adult skin healing is the unique ability of fetal wounds to heal without contracture and scar formation. Studies have shown that the ratio between the three isoforms of TGFbeta is different in adult and fetal wounds. Thus, we analyzed the capacity of adult and fetal human skin fibroblasts to contract collagen gels after stimulation with TGFbeta isoforms. In control medium, fetal fibroblasts had a contractile capacity similar to that of adult fibroblasts. However, the growth capacity of fetal fibroblasts was completely inhibited, in contrast to adult fibroblasts. When cells were treated with TGFbeta, fetal fibroblasts showed an inhibition of their contractile capacity whereas adult fibroblasts further contracted gels. The contractile response was similar for all isoforms of TGFbeta although TGFbeta3 always had the strongest effect. We considered that the regulation of cell contractile capacity by TGFbeta may be dependent on receptor expression for this cytokine, on myofibroblast differentiation of the cells, or in cell links with matrix. Since TGFbeta receptor analysis did not show differences in receptor affinity, we studied the expression of alpha-smooth muscle (SM) actin, a fibroblast contractile marker and of three integrins, the cell surface receptors specific of the attachment of the fibroblasts with collagen matrix. We observed that the expression of alpha-SM actin and alpha3 and beta1 integrin subunits was increased when TGFbeta was added to the medium of adult fibroblasts whereas the levels of the alpha1 and alpha2 subunits were unchanged. In contrast, fetal fibroblasts treated with TGFbeta showed a decrease of alpha1, alpha2, and beta1 integrin expression but no change in alpha3 integrin and in alpha-SM actin expression. These results indicate that intrinsic differences between fetal and adult fibroblasts might explain their opposite responses to TGFbeta stimuli. The variations in their alpha-SM actin and integrin expression patterns represent potentially important mechanisms used by fetal fibroblasts to regulate their response to cytokines, and likely contribute to the resultant differences in the quality of wound repair.

Actins↗

PubMed2001

Localization of specific binding sites for 125I-TGF-beta1 to fenestrated endothelium in bone and anastomosing capillary networks in enamel organ suggests a role for TGF-beta1 in angiogenesis.

Previous studies have shown endothelial cells to be a major target for endocrine TGF-beta in several soft tissues in the normal growing rat. The potent effect of TGF-beta1 on bone formation prompted us to analyze in detail the localization of specific binding sites for endocrine TGF-beta in hard tissues. At 2.5 minutes after injection of 125I-TGF-beta1, specific binding, as demonstrated by quantitative radioautography, was localized to fenestrated endothelium participating in angiogenesis in the vascular invasion region of the growth plate in bone as well as to anatomizing capillary networks in the maturation zone of the enamel organ. At 15 minutes after injection, the bound ligand was internalized into endocytic vesicles of endothelial cells. In bone, quantitation revealed significant differences in receptor density between endothelia undergoing proliferation vs those in a state of elongation and anastomosis with neighboring endothelial cells. In the rat incisor, specific binding of 125I-TGF-beta1 to endothelium correlated with increased formation of anastomotic capillary networks. These studies identify differential specific binding sites of 125I-TGF-beta1 in angiogenically active endothelium, providing an important link between TGF-beta1, the endothelium, and hard tissue development.

3T3 Cells↗

PubMed2001

Systemic steroid pretreatment improves cerebral protection after circulatory arrest.

BACKGROUND: This study evaluates whether systemic steroid pretreatment enhances neuroprotection during deep hypothermic circulatory arrest (DHCA) compared with steroid in cardiopulmonary bypass (CPB) prime. METHODS: Four-week-old piglets randomly placed into two groups (n = 5 per group) were given methylprednisolone (30 mg/kg) into the pump prime (group PP), or pretreated intravenously 4 hours before CPB (group PT). All animals underwent 100 minutes of DHCA (15 degrees C), were weaned off CPB, and were sacrificed 6 hours later. Postoperative changes in body weight, bioimpedance, and colloid oncotic pressure (COP) were measured. Cerebral trypan blue content, immunohistochemical evaluation of transforming growth factor-beta1 (TGF-beta1) expression, and caspase-3 activity were performed. RESULTS: Percentage weight gain (group PP 25.0% +/- 10.4% versus group PT 12.5% +/- 4.0%; p = 0.036), and percentage decrease in bioimpedance (PP 37.2% +/- 14.5% versus PT 15.6% +/- 7.9%; p = 0.019) were significantly lower, whereas postoperative COP was significantly higher in group PT versus group PP (PT 15.3 +/- 1.8 mm Hg versus PP 11.6 +/- 0.8 mm Hg; p = 0.003). Cerebral trypan blue (ng/g dry tissue) was significantly lower in group PT (PT 5.6 x 10(-3) +/- 1.1 x 10(-3) versus PP 9.1 x 10(-3) +/- 5.7 x 10(-4); p = 0.001). Increased TGF-beta1 expression and decreased caspase-3 activity were shown in group PT. CONCLUSIONS: Systemic steroid pretreatment significantly reduced total body edema and cerebral vascular leak and was associated with better immunohistochemical indices of neuroprotection after DHCA.

Animals↗

PubMed2001

Acquired Robertsonian translocations in two leukemia patients.

Robertsonian translocations were observed in two leukemia patients. The first case was a patient with chronic lymphocytic leukemia, who was found to have a rare Robertsonian translocation der(14;15)(q10;q10). The second case, a patient with acute myeloid leukemia, had multiple Robertsonian translocations: der(15)t(13;15)(q11.1;p11.1), der(14;22)(q10;q10), and dic(21;22)(p11.1;p11.1). Acquired multiple Robertsonian translocations have not been reported previously in leukemia.

Acute Disease↗

PubMed2000

Endoglin expression on human microvascular endothelial cells association with betaglycan and formation of higher order complexes with TGF-beta signalling receptors.

Transforming growth factor-beta (TGF-beta) plays an important role in angiogenesis and vascular function. Endoglin, a transmembrane TGF-beta binding protein, is highly expressed on vascular endothelial cells and is the target gene for the hereditary haemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. The specific function of endoglin responsible for HHT1 is believed to involve alterations in TGF-beta responses. The initial interactions on the cell surface between endoglin and TGF-beta receptors may be an important mechanism by which endoglin modulates TGF-beta signalling, and thereby responses. Here it is shown that on human microvascular endothelial cells, endoglin is co-expressed and is associated with betaglycan, a TGF-beta accessory receptor with which endoglin shares limited amino acid homology. This complex formation may occur in either a ligand-dependent or a ligand-independent manner. In addition, the occurrence of three higher order complexes containing endoglin, type II and/or type I TGF-beta receptors, on these cells is demonstrated. Our findings suggest that endoglin may modify TGF-beta signalling by interacting with both betaglycan and the TGF-beta signalling receptors at physiological receptor concentrations and ratios.

Affinity Labels↗

PubMed1999

Ectodomain cleavage and shedding of the type III transforming growth factor-beta receptor in lung membranes effect of temperature, ligand binding and membrane solubilization.

Previous studies from our laboratory [Philip, A. & O'Connor-McCourt, M. D. (1991) J. Biol. Chem. 266, 22290--22296] have shown that the lung exhibited the highest uptake of circulating [125I]-transforming growth factor-beta1 (TGF-beta1) on a per gram basis. This observation, together with the lack of information on TGF-beta receptor expression in the lung, prompted us to attempt to characterize TGF-beta receptors in this tissue. In the present report we show that the type III TGF-beta receptor is the most abundant TGF-beta binding protein in rat lung membranes and that it exhibits a 10-fold higher affinity for TGF-beta2 than for TGF-beta1. We observed that the majority of the type III receptor population in lung membranes is cleaved at a site in the central portion of the ectodomain, the resulting two fragments (95 kDa and 58 kDa) being held together by disulfide bonds. Furthermore, we demonstrate that a soluble form of the ectodomain of the type III receptor is shed from rat lung membranes in an efficient manner, with protease cleavage occurring at a site close to the transmembrane domain. This shedding is controllable by temperature, thus providing a system to study the mechanism of ectodomain release. Using this system, we show that the shedding is inhibited by prior ligand binding and by membrane solubilization. The identification of a membrane preparation which exhibits controllable and quantitative release of the type III receptor ectodomain provides a unique cell-free system for further studies of the mechanism of shedding of the type III TGF-beta receptor ectodomain.

Affinity Labels↗

PubMed1998

TGF-beta receptor expression on human keratinocytes: a 150 kDa GPI-anchored TGF-beta1 binding protein forms a heteromeric complex with type I and type II receptors.

Keratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-beta has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-beta receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-beta receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-beta1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-beta isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-beta receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-beta1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-beta receptors on keratinocytes, supporting the heterotetrameric model of TGF-beta signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-beta by markedly downregulating all four TGF-beta binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-beta1 binding protein forms a heteromeric complex with the TGF-beta signalling receptors suggests that this GPI-anchored protein may modify TGF-beta signalling in human keratinocytes.

Adult↗

PubMed1998

Transforming growth factor-beta receptor expression on human skin fibroblasts: dimeric complex formation of type I and type II receptors and identification of glycosyl phosphatidylinositol-anchored transforming growth factor-beta binding proteins.

Fibroblasts play a critical role in wound repair and in the development of fibrotic diseases, and transforming growth factor-beta (TGF-beta) has been shown to profoundly modulate fibroblast function. However, there is limited information on the TGF-beta receptor types, isoform specificity, and complex formation in skin fibroblasts. Here, we report that normal adult human skin fibroblasts display two isoform-specific, cell surface glycosyl phosphatidylinositol (GPI)-anchored, TGF-beta binding proteins in addition to the type I, II, and III TGF-beta receptors. The identities of these proteins are confirmed on the basis of their affinity for TGF-beta isoforms, immunoprecipitation with specific antireceptor antibodies, and other biochemical analyses. Immunoprecipitation results also indicated oligomeric complex formation between type I and II and between type II and III TGF-beta receptors. Furthermore, by using affinity labeling and two-dimensional electrophoresis, we demonstrate the occurrence of type I and II heterodimers and type I homodimers of TGF-beta receptors on these cells. Because the type I receptor does not bind TGF-beta in the absence of type II receptor, these results indicate that one molecule of TGF-beta induces the formation of a heterooligomeric complex containing more than one molecule each of type I and II TGF-beta receptors on these cells. These cells respond to TGF-beta by markedly down-regulating all five binding proteins and by potently augmenting DNA synthesis. These results allow the expansion of the proposed heteromeric TGF-beta receptor signaling paradigm using mutant cells that are unresponsive to TGF-beta and cell lines that have been transfected to overexpress these receptors, to include normal TGF-beta-responsive cells. In addition, the definition of TGF-beta receptor profiles in human skin fibroblasts provides important information for studying their alterations in these cells in various skin diseases.

Activin Receptors, Type I↗

PubMed1998

Angiogenesis and growth factor expression in a model of transmyocardial revascularization.

BACKGROUND: The mechanism by which transmyocardial revascularization (TMR) exerts a beneficial effect remains unknown. We hypothesize that the myocardial punctures of TMR cause a myocardial injury, leading to an angiogenic response mediated by a number of growth factors. METHODS: Fifty-three rats underwent ligation of the left coronary artery. Group I (n = 25) served as controls, whereas group II (n = 28) underwent concomitant TMR by the creation of six transmural channels with a 25-gauge needle in the ischemic zone. Surviving animals in both groups were sacrificed at intervals of 1, 2, 4, and 8 weeks (n = 5 in each subgroup). Immunohistochemistry in the infarct areas was performed for factor VIII to assess vascular density. Immunohistochemistry using specific antibodies was also performed for transforming growth factor-beta, basic-fibroblast growth factor, and vasoendothelial growth factor. Growth factor expression was quantitated by comparing areas of staining (in mm2) with computerized morphometric analysis. RESULTS: Mortality was similar in both groups (5/25 versus 8/28; not significant). Group II had significantly greater vascular density than group I (5.65 versus 4.06 vessels/high-power field; p < 0.001), with a peak at 1 week postoperatively (9.12 versus 5.56 vessels/high-power field; p < 0.0001) in both groups. Overall, levels of both transforming growth factor-beta and basic-fibroblast growth factor were significantly higher in the TMR group compared with the control group (0.207 versus 0.141 mm2/mm2, p < 0.05; and 0.125 versus 0.099 mm2/ mm2, p < 0.05). CONCLUSIONS: This model of TMR is associated with a significant angiogenic response, which appears to be mediated by the release of certain angiogenic growth factors such as transforming growth factor-beta and basic-fibroblast growth factor. With the long-term patency of laser-created myocardial channels in clinical TMR increasingly in doubt, its mechanism of myocardial revascularization may be similar to that observed in our model.

Animals↗

PubMed1998

Nonsteroidal-induced benign strictures of the colon: a case report and review of the literature.

Colonic strictures are a rare complication reported to result from chronic use of sustained release formulations of indomethacin and diclofenac. Such strictures often present with associated mucosal ulceration and are thought to result from nonsteroidal antiinflammatory drug-induced alterations in enterocyte homeostasis. Strictures generally occur in the cecum, ascending, and proximal transverse colon with symptoms of occult blood loss, obstruction, changes in bowel habits, and rarely, perforation. The first reported case of a 69-year-old woman who developed recurrent colonic strictures with inflammatory changes and mucosal ulceration while taking Lodine (etodolac) is presented. A brief review of the relevant literature and suggested preventative therapies are discussed.

Aged↗

PubMed1998

Pulmonary epithelial permeability and gas exchange: a comparison of inverse ratio ventilation and conventional mechanical ventilation in oleic acid-induced lung injury in rabbits.

STUDY OBJECTIVE: (1) To explore the interaction between mechanical ventilation and oleic acid (OA)-induced lung injury on indexes of pulmonary gas exchange and epithelial permeability, and (2) to compare this interaction using two different modes of ventilation: pressure-controlled inverse ratio ventilation (PCIRV) and volume-controlled ventilation with positive end-expiratory pressure (VCV PEEP). DESIGN: Randomized animal study. SETTING: Experimental laboratory investigation at Södersjukhuset, Stockholm, Sweden. ANIMALS: Twenty-four New Zealand white rabbits. INTERVENTIONS: (1) Ventilation with PCIRV (n=6) or VCV PEEP (n=6) for 6 h at equal end-expiratory alveolar pressure levels of 5 cm H2O followed by induction of lung injury (IV injection of OA 0.15 mL/kg). (2) Induction of lung injury followed by 6 h of ventilation with either PCIRV (n=6) or VCV PEEP (n=6) as described above. MEASUREMENTS AND RESULTS: Lung mechanics, heart rate, BP, and gas exchange results were equal at baseline. In group A, after 1 h of ventilation, mean airway pressure was 11.9+/-4.4 with PCIRV and 8.3+/-1.0 cm H2O with VCV PEEP (p<0.05). Forty minutes after OA injection, PaO2/fraction of inspired oxygen (FIO2) was 24+/-10 kPa with PCIRV and 44+/-15 kPa with VCV PEEP (p<0.05). Mean airway pressure was higher and peak airway pressure was lower with PCIRV. In group B, after 6 h of ventilation, PaO2/FIO2 was 17+/-5 kPa with PCIRV and 43+/-8 kPa with VCV PEEP (p<0.01). Systemic BP was lower with PCIRV and mean airway pressure was higher. Technetium-99m diethylene triamine penta-acetic acid lung clearance: In group A, curves were monoexponential with both PCIRV (half-life time [T 1/2], 21+/-8 min and VCV PEEP (T 1/2, 126+/-59 min, p<0.005) until injection of OA. In the VCV PEEP-treated animals, a marked increase in clearance rate was observed within 60 s of OA injection (T 1/2, 13+/-9 min, p<0.001). Fifteen minutes after OA injections, T 1/2 had decreased to 38+/-17 min with VCV PEEP. In the animals treated with PCIRV, OA injection did not lead to a significant change in clearance rate, although the elimination pattern was observed to change from single-compartment to multicompartment type. In group B, clearance curves were monoexponential with both ventilatory modes. There was no significant difference in clearance rate between PCIRV (T 1/2, 25+/-9 min) and VCV PEEP (T 1/2, 36+/-16 min, not significant). CONCLUSIONS: The observation that PaO2 was lower in the PCIRV-treated groups must be interpreted with caution in this animal study with relatively few observations. The finding may reflect differences in the effect of OA injection in the two ventilatory modes. It is also possible that externally applied PEEP is more effective than PCIRV in increasing oxygen tension, either because of a less inhomogenous distribution of ventilation and perfusion or for other reasons. The clearance results imply that PCIRV causes an alteration in lung epithelial or membrane function in comparison to VCV PEEP. This functional difference is most likely caused by the large time-weighted lung volume produced by pressure control in combination with a prolonged inspiration. Induction of high permeability lung injury with OA eliminates the difference between PCIRV and VCV PEEP. It remains to be established whether these findings are relevant with regard to ventilator-associated structural lung injury in man.

Acute Disease↗

PubMed1996

Pulmonary epithelial permeability. An animal study of inverse ratio ventilation and conventional mechanical ventilation.

STUDY OBJECTIVE: To compare pressure-controlled inverse ratio ventilation (PCIRV) with volume-controlled ventilation with positive end-expiratory pressure (VCV PEEP) at equal levels of end-expiratory alveolar pressure. The primary focus of the study was on pulmonary epithelial permeability. Histologic and gravimetric indicators of lung injury were also studied. DESIGN: Randomized animal study. SETTING: Experimental investigation at Södersjukhuset, Stockholm, Sweden. ANIMALS: Thirty-two New Zealand white rabbits. INTERVENTIONS: Ventilation with PCIRV or VCV PEEP for 6 h at an end-expiratory pressure level of 5 cm H2O. MEASUREMENTS AND RESULTS: Lung mechanics, heart rate, BP, and gas exchange. Measurement of pulmonary epithelial permeability by 99mTc-DTPA lung clearance. Extravascular lung water by gravimetric analysis. Morphology by light microscopy after a perfusion fixation procedure. Mean and peak airway pressures were 12.4 +/- 4.3 and 15.9 +/- 4.5 cm H2O with PCIRV and 8.6 +/- 0.8 (p < 0.001) and 19.9 +/- 4.1 cm H2O (p < 0.03) with VCV PEEP at 6 h. Mean systemic BP was lower with PCIRV (58 +/- 9 mm Hg) than with VCV PEEP (68 +/- 7 mm Hg) at 6 h (p < 0.003). At 6 h, PaCO2 was lower with PCIRV (3.2 +/- 0.6 kPa) than with VCV PEEP (4.1 +/- 0.8 kPa) (p < 0.02). There was no difference in blood oxygenation between PCIRV and VCV PEEP. 99mTc-DTPA lung clearance curves were monoexponential with both PCIRV and VCV PEEP. Mean lung clearance expressed as T 1/2 was 16 +/- 9 min with PCIRV and 107 +/- 74 min with VCV PEEP (p < 0.001). Morphologic examination revealed no differences between the groups and no evidence of significant lung injury. CONCLUSIONS: The observations reported in this article imply that PCIRV causes an alteration in lung epithelial or membrane function in comparison to VCV PEEP. This functional difference is most likely caused by the large time-adjusted lung volume produced by pressure control in combination with a prolonged inspiration. It remains to be established whether this early functional effect of PCIRV is relevant with regard to structural lung injury in mechanically ventilated subjects.

Animals↗

PubMed1995

v-Myb DNA binding is required to block thrombocytic differentiation of Myb-Ets-transformed multipotent haematopoietic progenitors.

The E26 avian leukaemia virus encodes a fusion oncoprotein consisting of truncated versions of the c-Myb and c-Ets-1 transcription factors. When used to infect embryonic chicken haematopoietic cells two types of self-renewing progenitors are obtained, namely myeloblasts and 'MEPs' (Myb-Ets progenitors). In earlier work we have shown that myeloblasts transformed by the ts21 mutant of E26, which has a lesion in v-Myb, can be induced to differentiate into macrophages following shift to the non-permissive temperature. Here we show that the ts21 v-Myb is temperature sensitive for DNA binding in band shift experiments and that its inactivation in transformed MEPs induces their maturation into thrombocytes. The MEP transforming capacity of v-Myb is not confined to its fusion with v-Ets, as it is also seen with a virus that co-expresses tsMyb with v-ErbB. As with wild-type E26-transformed MEPs, ts21-transformed MEPs are multipotent, differentiating into eosinophils and myeloblasts following treatment with 12-O-tetradecanoylphorbol-13-acetate. In addition, ts21-transformed myeloblasts differentiate into macrophages when shifted to the non-permissive temperature. This shows that v-Myb blocks haematopoietic differentiation at two distinct stages. In contrast, v-Ets inactivation in MEPs transformed by a ts E26 mutant with a lesion in the corresponding oncoprotein leads to their differentiation into erythrocytes, myeloblasts and probably eosinophils. These data show that the two domains of Myb-Ets selectively affect decision making processes in different types and stages of haematopoietic cells.

Acetyltransferases↗

PubMed1995

Transforming growth factor-beta receptors on human endometrial cells: identification of the type I, II, and III receptors and glycosyl-phosphatidylinositol anchored TGF-beta binding proteins.

In the present study, we have characterized the cell surface receptors for transforming growth factor-beta (TGF-beta) on monolayer cultures of stromal cells prepared from human endometrial biopsies, and on a human endometrial epithelial cell line (RL95-2) using affinity cross-link labeling techniques. On the stromal cells, five TGF-beta binding proteins were identified. Analysis of the sensitivity of these proteins to dithiothreitol and phosphatidylinositol-specific phospholipase C, together with results from immunoprecipitations with antibodies against the type II and III TGF-beta receptors, confirmed that three of these binding proteins correspond to the cloned type I, II, and III TGF-beta receptors. The other two binding proteins observed exhibit the characteristics of isoform-specific GPI-anchored TGF-beta binding proteins. On RL95-2 cells, three TGF-beta binding proteins, corresponding to the type I, II, and III TGF-beta receptors, were identified. The receptors which we have characterized on endometrial cells are responsive to physiological concentrations of TGF-beta as demonstrated by the effect of TGF-beta on endometrial cell proliferation. Accordingly, these receptors have the potential to respond to the TGF-beta isoforms which have recently been detected in the endometrium in an autocrine and/or paracrine manner.

Affinity Labels↗

PubMed1995

Characterization of transforming growth factor-beta growth regulatory effects and receptors on bovine mammary cells.

Transforming growth factor-beta (TGF-beta) has been shown to inhibit mammary morphogenesis, growth, and differentiation in murine studies. We have characterized TGF-beta receptors and their autoregulation, and the growth response to TGF-beta 1 and TGF-beta 2 in cultured bovine mammary epithelium (MAC-T) and fibroblasts. Affinity labelling studies revealed that fibroblast and epithelial cells contained type I, II, and III (betaglycan) receptors, with the type III receptor being the predominant binding component. On both fibroblasts and epithelial cells, TGF-beta 1 and TGF-beta 2 had equal binding affinities for the type I and II receptors, but TGF-beta 2 had a higher affinity for the type III receptor. Also, preincubation of MAC-T cells with 50 pM TGF-beta 1 or TGF-beta 2 markedly downregulated TGF-beta receptors. Proliferative response was measured using both total DNA and 3H-thymidine incorporation. Both TGF-beta isoforms were effective in inhibiting proliferation of MAC-T cells and fibroblasts. Inhibition of proliferation was not altered following immortalization of fibroblasts with SV-40 Large-T-antigen (LT), even when the cells acquired a transformed phenotype. Inhibition of proliferation was not a result of cytotoxicity, as TGF-beta at concentrations 1,000-fold higher than ED50 levels did not increase cell death. Moreover, the inhibition was reversible as shown by return of cellular proliferation to control levels following TGF-beta removal. Although growth inhibition was not transient as culture of MAC-T cells in TGF-beta resulted in sustained inhibition of proliferation for at least 144 h.

Animals↗

PubMed1995

Specific binding of endocrine transforming growth factor-beta 1 to vascular endothelium.

The presentation of recombinant biologically active 125I-TGF-beta 1 via the bloodstream to potential target cells in mice and rats was evaluated by quantitative light and electron microscope radioautography. Specificity was evaluated by in vivo competition with excess unlabeled TGF-beta 1, and integrity of the ligand at the binding site was demonstrated by trichloroacetic acid precipitation after extraction from tissues. The distribution of radiolabel at 2.5, 15, 30, 45, and 60 min after 125I-TGF-beta 1 injection revealed radiolabel principally over microvasculature endothelium but at times > 2.5 min over endothelial endocytic components indicative of internalization. Nonspecific binding of 125I-TGF-beta 1 to the apex of the proximal convoluted tubule of the kidney indicated it as the likely site of rapid clearance of TGF-beta 1 from the circulation, while a comparison of the binding of 125I-TGF-beta 1 (endothelial) to that of 125I-TGF-beta 1 complexed with alpha 2-macroglobulin-methylamine (liver parenchyma) indicated that clearance of TGF-beta 1 complexed alpha 2-macroglobulin was likely via the hepatic alpha 2-macroglobulin receptor. The endothelial TGF-beta receptors uncovered here are likely involved in the local regulatory mechanism of leukocyte and monocyte adhesion and tissue infiltration regulated by endocrine TGF-beta 1.

Animals↗