摘要:

AbstractThe site of action of cysteine‐proteinases (CPs) and matrix metalloproteinases (MMPs) in the degradation of bone collagen by osteoclasts was investigated by evaluating the effects of the CP‐inhibitor trans‐epoxy‐succinyl‐L‐leucylamido (4‐guanidino)‐butane (E‐64) and the MMP‐inhibitor N‐(3‐N‐benzyloxycarbonyl amino‐1‐R‐carboxypropyl)‐L‐leucyl‐O‐methyl‐L‐tyrosine N‐methylamide (Cl‐1) in an in vitro model system of PTH‐stimulated mouse calvaria. In the presence of each of the two inhibitors a large area of collagen free of mineral crystallites was seen adjacent to the ruffled border of the osteoclasts. Following a culture period of 24 h this area proved to be about 10 times larger in inhibitor‐treated explants than in controls. Moreover the percentage of osteoclasts in close contact with such demineralized bone areas appeared to be significantly higher in inhibitor‐treated explants than in control specimens (60% and 5%, respectively). These effects were not apparent when the osteoclastic activity was inhibited with calcitonin. No significant differences were found between the effects of the two inhibitors, E‐64 and Cl‐1. Our observations indicate that under the influence of inhibitors of MMPs and CPs demineralization of bone by osteoclasts proceeded up to a certain point whereas matrix degradation was strongly inhibited. It is concluded that within the osteoclastic resorption lacuna both CPs and MMPs part

ISSN:0021-9541    

DOI:10.1002/jcp.1041500202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have studied the effect of transforming growth factor β1 (TGF‐β1) on vascular smooth muscle cell (SMC) mitogenesis and expression of thrombospondin and other growth related genes. We found that TGF‐β1 treatment of vascular SMC induced a prolonged increase in steady‐state mRNA levels of thrombospondin as well as α1(lV) collagen. The increase began at approximately 2 h, peaked by 24 h, and remained considerably elevated 48 h after growth factor addition. There was a corresponding increase in thrombospondin protein as well as increased expression of several other secreted polypeptides. The increase in thrombospondin contrasted sharply with that observed for platelet‐derived growth factor (PDGF) which induced a rapid and transient increase in thrombospondin mRNA level. Although TGF‐β1 was able to directly enhance expression of thrombospondin as well as the growth‐related genes c‐fos and c‐myc, and induced c‐fos expression with identical kinetics as PDGF, it was unable to elicit [3H]thymidine incorporation into DNA in three independent smooth muscle cell strains. However, TGF‐β1 was able to strongly increase the mitogenic response of SMC to PDGF. Addition of both TGF‐β1 and PDGF to SMC also caused a synergistic increase in the expression of thrombospondin as well as c‐myc. Interestingly, in one other smooth muscle cell strain, a weak and delayed mitogenic response to TGF‐β1 alone was observed. Our results strongly suggest that induction of throm‐bospondin expression by TGF‐β1 and by PDGF occurs by distinct mechanisms. In addition, that TGF‐β1 can enhance PDGF‐induced mitogenesis may be due to the ability of TGF‐β1 to directly induce the expression of thrombo

ISSN:0021-9541    

DOI:10.1002/jcp.1041500203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe properties of primary rabbit kidney proximal tubule cells in glucose‐free serum‐free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose‐free and in glucose‐supplemented medium. Growth in glucose‐free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose‐free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose‐free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D‐glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose conten

ISSN:0021-9541    

DOI:10.1002/jcp.1041500204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe basal lamina protein, laminin, has been shown to promote migration and proliferation of cultured skeletal myoblasts, resulting in increased myotube formation. However, skeletal myotubes adhere poorly to a laminin substrate, and long‐term cultures of skeletal myotubes on laminin have not been achieved. We have found that cultured satellite cells from bupivacaine‐damaged rat skeletal muscle actively proliferate and differentiate on a diluted Matrigel substrate composed of laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. Myotubes cultured on diluted Matrigel are contractile and have never been observed to detach from the culture dish; rather, myotubes generally atrophy after 2–3 weeks in culture. Antibodies directed against the various protein components of Matrigel were used to determine the role of each component in enhancing muscle differentiation. Anti‐laminin impaired satellite cell adhesion, whereas antibodies against either type IV collagen or heparan sulfate proteoglycan had no effect. Anti‐entactin did not inhibit attachment, proliferation, or fusion of cultured satellite cells; however, myotubes exposed to anti‐entactin failed to adhere to the culture dish after spontaneous myotube contractions began. We conclude that entactin is responsible for long‐term maintenance and maturation of contractile skeletal myotubes on a diluted Matrigel substrate. This is the first study to assign a biological function for entactin

ISSN:0021-9541    

DOI:10.1002/jcp.1041500205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe dependence of urokinase‐type plasminogen activator (uPA) induction on endogenous basic fibroblast growth factor (bFGF) activity during endothelial cell migration was investigated utilizing a combination of wounded endothelial cell monolayers and substrate overlay techniques. Purified polyclonal rabbit immunoglobulin G (IgG) against bFGF blocked the appearance of uPA‐dependent lytic activity normally observed at the edge of a wounded bovine aortic endothelial (BAE) cell monolayer. Additionally, the migration of cells into the denuded area was inhibited 30–50% by antibodies either to bFGF or to bovine uPA. Incubation of wounded monolayers with either purified bovine uPA or agents able to induce PA activity, such as phorbol myristate acetate (PMA), vanadate, or bFGF, resulted in enhanced migration of cells (28–50%). Anti‐bovine uPA IgG blocked a significant fraction (25%) of BAE cell migration induced by exposure to exogenous bFGF. The role of uPA in migration of wounded BAE cells was not dependent on plasmin generation. Furthermore, the amino terminal fragment (ATF) of human recombinant (hr) uPA, which is enzymatically inactive, stimulated BAE cell movement (36%) as well as intact uPA. ATF of hr uPA also stimulated endothelial cell movement in the presence of anti‐bFGF IgG. These results suggest that BAE cell migration from the edge of a wounded monolayer is dependent upon local increases of uPA mediated by endogenous bFGF. Moreover, the data support the conclusion that migration is stimulated via a signalling mechanism dependent upon occupancy of the uPA receptor but independent of uPA‐mediate

ISSN:0021-9541    

DOI:10.1002/jcp.1041500206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN‐γ). We studied the influence of keratinocyte mitogens such as transforming growth factor‐alpha (TGF‐α), epidermal growth factor (EGF), and somatomedin‐C (SM‐C) on the ligand binding of32P‐labelled IFN‐γ to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN‐γ receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF‐α (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN‐γ receptor sites by TGF‐α/EGF was mitogen specific, as adding SM‐C (500 ng/ml) did not have any effect on ligand binding, although it similarily stimulated keratinocyte growth. The reduction in IFN‐γ receptors was time dependent, occurring primarily after 24–48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN‐γ receptors by TGF‐α/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA‐DR and intercellular adhesion molecule‐1 (ICAM‐1) induced by IFN‐γ, compared to actively growing TGF‐α/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF‐α/EGF but not SM‐C are capable of altering their response to IFN‐γ by decreasing t

ISSN:0021-9541    

DOI:10.1002/jcp.1041500207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe predominant cyclooxygenase products of keratinocytes are prostaglandin (PG)E2and PGF2αwith only trace amounts of PGI2synthesis detected. When normal or immortal (NM1) keratinocytes were co‐cultured with mitomycin C‐treated 3T3 cells, increased synthesis of PGI2was noted compared to mitomycin C‐treated 3T3 cells alone. The PGI2level in co‐cultures was maximum within the first week and diminished rapidly thereafter. These results suggested keratinocytes enhance the production of PGI2by 3T3 cells. Keratinocyte cultures incubated with lloprost and Piriprost, stable PGI2analogues, showed evidence of increased cornification as demonstrated by staining with rhodanile blue, decreased shedding of cells into the culture medium, and more cornified material adhering to the culture surface. The cultures appeared to be responsive between the first and second weeks after plating and the inhibition of shedding could not be reversed by changing to drug‐free medium. Control and treated cultures showed identical electrophoretic protein patterns. Immunoblots showed involucrin unchanged in extracts of control and treated cultures while the 22 kd pancornulin was absent in treated cultures. The findings that keratinocytes enhance the production of PGI2by 3T3 cells and that PGI2analogues enhance cornification of confluent keratinocytes raise the possibility that eicosanoids may serve as autoregulatory signals together with oth

ISSN:0021-9541    

DOI:10.1002/jcp.1041500208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThis study was designed to examine the state of proliferation in the rat thyrocyte following the administration of thyroid stimulating hormone (TSH). An immunohistochemical technique involving the use of a monoclonal antibody to statin, a nonproliferation‐specific nuclear antigen, was developed to measure the subpopulation of cells that have ceased to divide. Following the random assignment of young male Sprague‐Dawley rats into various groups, the rats in the control group received a single intraperitoneal (i‐p) injection of normal saline, whereas the experimental groups received single i‐p injections of TSH at doses of 0.25, 0.50, and 1.0 IU, respectively. All rats were subsequently sacrificed in groups of three at 1, 2, 4, and 24 hours. The statin antibody label was readily identified within the follicle cell nucleus. Results revealed a statistically significant transient decrease in the mean percent statin‐positive nuclei in the TSH‐treated groups. The time‐ and dose‐dependent effect of TSH was maximal at 2 hours and no longer discernible at 24 hours. A second experiment involving the chronic administration of TSH (i‐p 0.25 IU twice daily) resulted in a cumulative response with a statistically significant progressive decrease in the mean percent of statin‐positive nuclei at 5 and 10 days, returning to near normal values 5 days following the cessation of treatment. Determination of the nuclear optical density of the statin reaction product by image analysis techniques revealed that a single injection of TSH resulted in a rapid disappearance of the statin nuclear protein. This result suggests that the disappearance of statin in the nucleus appears to reflect the event of cells leaving the nondividing quiescent state to resume the cell cycle traverse following the administration of TSH. The disappearance of statin appears as an early nuclear event that parallels the earliest known cytoplasmic pinocytotic response to TSH in the rat th

ISSN:0021-9541    

DOI:10.1002/jcp.1041500209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA conjugate of horseradish peroxidase (HRP) to poly(L‐lysine) (PLL) was used to characterize a non‐lysosomal proteolytic compartment in the MDCK Strain I epithelial cell line. This compartment is expressed in a polar fashion, and is capable of degradation of the PLL moiety in the conjugate followed by release of HRP via a basal‐to‐apical, but not apical‐to‐basal, transcytotic pathway. This uptake, cleavage, and transport process appears to require approximately 2 hr, as there is a 2 hr lag‐time between conjugate administration to the basal surface and HRP release to the apical medium. Monensin (10 μM) failed to inhibit this process, indicating that participation of the trans‐Golgi network (TGN) in the trafficking of internalized conjugate is not the rate‐determining step. Inhibition of HRP transport was found to be elicited by 50 μg/ml leupeptin, but only when applied to the basal surface. Brief trypsinization of either the basal or apical surfaces of cells preloaded with HRP conjugate showed no appreciable inhibitory effect on the apical release of HRP, indicating that an intracellular compartment rather than surface‐bound enzymes is responsible for the degradation of the PLL moiety in the conjugate. Our results demonstrate the presence of an intracellular proteolytic compartment which is accessible in the basal‐to‐apical, but not apical‐to‐basal, transport pathway; and this compartment can be exploited for the transcytosi

ISSN:0021-9541    

DOI:10.1002/jcp.1041500210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThis study was undertaken to gain more insight into the mechanism whereby TGF‐β influences the cell cycle progression of cultured rabbit articular chondrocytes. Using proliferating chondrocytes in fetal calf serum‐containing medium, we have previously shown that TGF‐β induced a recruitment of cells at the end of the S phase (G2/M) observed 24 h after addition. The delayed cells may then be released, producing a proliferative effect at 48 h, provided a substantial amount of FCS (10%) is present in the medium. Otherwise, in low level of serum (2% FCS, for example), only inhibition of cell proliferation is observed. In chondrocytes synchronized in S phase by a thymidine block, we investigated here the time‐course incorporation of [3H]‐thymidine into DNA, the cell cycle traverse by flow cytofluorometric study of DNA content, the expression of PCNA (Proliferating Cell Nuclear Antigen), and cAMP levels. The data demonstrate that TGF‐β provoked a decrease of cAMP content (0.5–1 h) followed by an enhancement of the DNA synthesis rate (4 h) which was detectable through cytofluorometric analysis and [3H]‐thymidine labeling and correlated with the PCNA expression. In contrast, addition of cAMP analogues to the cultures resulted in an inhibition of replication rate. We also showed that pertussis toxin produced a decrease of the DNA synthesis rate, in a transient manner and only in the presence of TGF‐β. All these results suggest that TGF‐β may accelerate the replication process of cyclized chondrocytes, making then accumulate at the G2/M boundary, via a mechanism that could involve the adenylate cyclase activity and a Gi‐protein. The factor might be responsible for producing a pool of cells having already replicated their DNA and therefore capable of re‐entering the cell cycle without delay. This cell population could serve as a tissue reserve able to induce a mitosis wave when necessary—for example,

ISSN:0021-9541    

DOI:10.1002/jcp.1041500211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY