Flexor tendon wound healing in vitro: lactate up-regulation of TGF-beta expression and functional activity

Flexor tendon wound healing in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. These adhesions can significantly alter tendon gliding and ultimately hand function. Lactate and transforming growth factor-beta (TGF-beta) are two important mediators of wound healing that have been demonstrated to independently increase collagen production by cells of the tendon sheath, epitenon, and endotenon. This study examined the effects of lactate on TGF-beta peptide and receptor production by flexor tendon cells. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. Cell cultures were supplemented with 50 mM lactate, and the expression of three TGF-beta peptide isoforms (beta1, beta2, and beta3) and three receptor isoforms (R1, R2, and R3) was quantified with enzyme-linked immunosorbent assays. TGF-beta functional activity was also assessed with the addition of tendon cell conditioned media to mink lung epithelial cells transfected with a luciferase reporter gene expression construct responsive to TGF-beta. Supplementation of the cell culture medium with lactate significantly (p < 0.05) increased the expression of all TGF-beta peptide and receptor isoforms in all three cell lines. Tendon sheath fibroblasts exhibited the greatest increases in beta1 and beta2 peptide isoform expression (30 and 23 percent, respectively), whereas endotenon tenocytes demonstrated the greatest increase in beta3 peptide expression (32 percent). Epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression (17 and 19 percent, respectively). All three tendon cell types demonstrated significant (p < 0.05) increases in TGF-beta functional activity when exposed to lactate. Epitenon tenocytes demonstrated the greatest increase in activity (>4 times control values), whereas tendon sheath fibroblasts demonstrated the highest overall levels of total TGF-beta functional activity. Lactate significantly increased TGF-beta peptide (beta1, beta2, and beta3) expression, receptor (R1, R2, and R3) expression, and functional activity, suggesting a common pathway regulating tendon cell collagen production. Modulation of lactate and TGF-beta levels may provide a means of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.     

Down-regulation of transforming growth factor-beta receptors: cooperativity between the types I, II, and III receptors and modulation at the cell surface.

The types I, II, and III receptors (RI, RII, RIII) for transforming growth factor-beta (TGF-beta) become down-regulated in response to ligand, presumably via their internalization from the cell surface. This report examines the down-regulation of full-length RI, RII, and RIII in cells endogenously or transiently expressing these receptors. Down-regulation occurred rapidly (within 2 h after TGF-beta1 treatment at 37 degrees C) and showed a dose response, between 10 and 200 pM TGF-beta1, in cells expressing RI, RII, and RIII (Mv1lu and A549 cells). A comparison between Mv1Lu and mutant cell derivatives R-1B (lacking RI) or DR-26 (lacking RII) indicated that all three receptors were necessary for efficient down-regulation. Down-regulation experiments, utilizing TGF-beta-treated 293 cells transiently expressing different combinations of these receptors indicated that neither RII or RIII were down-regulated when expressed alone and that RI was required for maximal down-regulation of RII. RII and RIII were partially down-regulated when these receptors were coexpressed in the absence of RI (in R-1B and 293 cells). Surprisingly, TGF-beta receptors were partially down-regulated in Mv1Lu, A549, and 293 cells treated with TGF-beta1 at 4 degrees C. Microscopic examination of 293 cells coexpressing RI fused to green fluorescent protein (RI-GFP) and RII indicated that, after treatment with TGF-beta1 at 4 degrees C, RI-GFP formed aggregates at the cell surface at this temperature. RI-GFP was not detected at the surface of these cells after TGF-beta1 treatment at 37 degrees C. Our results suggest a two phase mechanism for TGF-beta1 receptor down-regulation involving receptor modulation (aggregation) at the cell surface and internalization.     

Studies in flexor tendon wound healing: neutralizing antibody to TGF-beta1 increases postoperative range of motion

The postoperative outcome of hand flexor tendon repair remains limited by tendon adhesions that prevent normal range of motion. Recent studies using in situ hybridization techniques have implicated transforming growth factor beta-1 (TGF-beta1) in both intrinsic and extrinsic mechanisms of repair. TGF-beta1 is a growth factor that plays multiple roles in wound healing and has also been implicated in the pathogenesis of excessive scar formation. The purpose of this study was to examine the effect of neutralizing antibody to TGF-beta1 in a rabbit zone II flexor tendon wound-healing model. Twenty-two adult New Zealand White rabbits underwent complete transection of the middle digit flexor digitorum profundus tendon in zone II. The tendons were immediately repaired and received intraoperative infiltration of one of the following substances: (1) control phosphate-buffered saline; (2) 50 microg neutralizing antibody to TGF-beta1; (3) 50 microg each of neutralizing antibody to TGF-beta1 and to TGF-beta2. Eight rabbits that had not been operated on underwent analysis for determination of normal flexion range of motion at their proximal and distal interphalangeal joints, using a 1.2-N axial load applied to the flexor digitorum profundus tendon. All rabbits that had been operated on were placed in casts for 8 weeks to allow maximal tendon adhesion and were then killed to determine their flexion range of motion. Statistical analysis was performed using the Student's unpaired t test. When a 1.2-N load was used on rabbit forepaws that had not been operated on, normal combined flexion range of motion at the proximal and distal interphalangeal joints was 93+/-6 degrees. Previous immobilization in casts did not reduce the range of motion in these forepaws (93+/-4 degrees). In the experimental groups, complete transection and repair of the flexor digitorum profundus tendon with infiltration of control phosphate-buffered saline solution resulted in significantly decreased range of motion between the proximal and distal phalanges [15+/-6 degrees (n = 8)]. However, in the tendon repairs infiltrated with neutralizing antibody to TGF-beta1, flexion range of motion increased to 32+/-9 degrees (n = 7; p = 0.002). Interestingly, a combination of neutralizing antibody to TGF-beta1 and that to TGF-beta2 did not improve postoperative range of motion [18+/-4 degrees (n = 7; p = 0.234)]. These data demonstrate that (1) the rabbit flexor tendon repair model is useful for quantifying tendon scar formation on the basis of degrees of flexion between proximal and distal phalanges; (2) intraoperative infiltration of neutralizing antibody to TGF-beta1 improves flexor tendon excursion; and (3) simultaneous infiltration of neutralizing antibody to TGF-beta2 nullifies this effect. Because TGF-beta1 is thought to contribute to the pathogenesis of excessive scar formation, the findings presented here suggest that intraoperative biochemical modulation of TGF-beta1 levels limits flexor tendon adhesion formation.     

Endocytosis of the type III transforming growth factor-beta (TGF-beta) receptor through the clathrin-independent/lipid raft pathway regulates TGF-beta signaling and receptor down-regulation

Transforming growth factor-beta (TGF-beta) signals through three highly conserved cell surface receptors, the type III TGF-beta receptor (T beta RIII), the type II TGF-beta receptor (T beta RII), and the type I TGF-beta receptor (T beta RI) to regulate diverse cellular processes including cell proliferation, differentiation, migration, and apoptosis. Although T beta RI and T beta RII undergo ligand-independent endocytosis by both clathrin-mediated endocytosis, resulting in enhanced signaling, and clathrin-independent endocytosis, resulting in receptor degradation, the mechanism and function of T beta RIII endocytosis is poorly understood. T beta RIII is a heparan sulfate proteoglycan with a short cytoplasmic tail that functions as a TGF-beta superfamily co-receptor, contributing to TGF-beta signaling through mechanisms yet to be fully defined. We have reported previously that T beta RIII endocytosis, mediated by a novel interaction with beta arrestin-2, results in decreased TGF-beta signaling. Here we demonstrate that T beta RIII undergoes endocytosis in a ligand and glycosaminoglycan modification-independent and cytoplasmic domain-dependent manner, with the interaction of Thr-841 in the cytoplasmic domain of T beta RIII with beta-arrestin2 enhancing T beta RIII endocytosis. T beta RIII undergoes both clathrin-mediated and clathrin-independent endocytosis. Importantly, inhibition of the clathrin-independent, lipid raft pathway, but not of the clathrin-dependent pathway, results in decreased TGF-beta1 induced Smad2 and p38 phosphorylation, supporting a specific role for clathrin-independent endocytosis of T beta RIII in regulating both Smad-dependent and Smad-independent TGF-beta signaling.     

Transforming growth factor (TGF)-beta 1 internalization: modulation by ligand interaction with TGF-beta receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis

Transforming growth factor-beta (TGF-beta) internalization was studied by monitoring the uptake of (125)I-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-beta receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degrees C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with (125)I- TGF-beta1 at 37 degrees C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degrees C for 2 h prior to switching the cells to 37 degrees C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degrees C and by a reduction in the labeling intensities of RI and RII in (125)I-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degrees C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degrees C, suggesting that prebinding promotes formation of stable RI.RII complexes that can signal independently of ligand.     

Induction of Transforming Growth Factor Beta Receptors following Focal Ischemia in the Rat Brain

Transforming growth factor-βs (TGF-βs) regulate cellular proliferation, differentiation, and survival. TGF-βs bind to type I (TGF-βRI) and II receptors (TGF-βRII), which are transmembrane kinase receptors, and an accessory type III receptor (TGF-βRIII). TGF-β may utilize another type I receptor, activin-like kinase receptor (Alk1). TGF-β is neuroprotective in the middle cerebral artery occlusion (MCAO) model of stroke. Recently, we reported the expression pattern of TGF-β1-3 after MCAO. To establish how TGF-βs exert their actions following MCAO, the present study describes the induction of TGF-βRI, RII, RIII and Alk1 at 24 h, 72 h and 1 mo after transient 1 h MCAO as well as following 24 h permanent MCAO using in situ hybridization histochemistry. In intact brain, only TGF-βRI had significant expression: neurons in cortical layer IV contained TGF-βRI. At 24 h after the occlusion, no TGF-β receptors showed induction. At 72 h following MCAO, all four types of TGF-β receptors were induced in the infarct area, while TGF-βRI and RII also appeared in the penumbra. Most cells with elevated TGF-βRI mRNA levels were microglia. TGF-βRII co-localized with both microglial and endothelial markers while TGF-βRIII and Alk1 were present predominantly in endothels. All four TGF-β receptors were induced within the lesion 1 mo after the occlusion. In particular, TGF-βRIII was further induced as compared to 72 h after MCAO. At this time point, TGF-βRIII signal was predominantly not associated with blood vessels suggesting its microglial location. These data suggest that TGF-β receptors are induced after MCAO in a timely and spatially regulated fashion. TGF-β receptor expression is preceded by increased TGF-β expression. TGF-βRI and RII are likely to be co-expressed in microglial cells while Alk1, TGF-βRII, and RIII in endothels within the infarct where TGF-β1 may be their ligand. At later time points, TGF-βRIII may also appear in glial cells to potentially affect signal transduction via TGF-βRI and RII.     

The cell biology of suturing tendons

Trauma by suturing tendon form areas devoid of cells termed “acellular zones” in the matrix. This study aimed to characterise the cellular insult of suturing and acellular zone formation in mouse tendon. Acellular zone formation was evaluated using single grasping sutures placed using flexor tendons with time lapse cell viability imaging for a period of 12 h. Both tension and injury were required to induce cell death and cell movement in the formation of the acellular zone. DNA fragmentation studies and transmission electron microscopy indicated that cells necrosed.Parallel in vivo studies showed that cell-to-cell contacts were disrupted following grasping by the suture in tensioned tendon. Without tension, cell death was lessened and cell-to-cell contacts remained intact. Quantitative immunohistochemistry and 3D cellular profile mapping of wound healing markers over a one year time course showed that acellular zones arise rapidly and showed no evidence of healing whilst the wound healing response occurred in the surrounding tissues. The acellular zones were also evident in a standard modified “Kessler” clinical repair. In conclusion, the suture repair of injured tendons produces acellular zones, which may potentially cause early tendon failure.     

Down-regulation of Transforming Growth Factor-β Receptors: Cooperativity between the Types I, II, and III Receptors and Modulation at the Cell Surface

The types I, II, and III receptors (RI, RII, RIII) for transforming growth factor-β (TGF-β) become down-regulated in response to ligand, presumably via their internalization from the cell surface. This report examines the down-regulation of full-length RI, RII, and RIII in cells endogenously or transiently expressing these receptors. Down-regulation occurred rapidly (within 2 h after TGF-β1 treatment at 37°C) and showed a dose response, between 10 and 200 pM TGF-β1, in cells expressing RI, RII, and RIII (Mv1lu and A549 cells). A comparison between Mv1Lu and mutant cell derivatives R-1B (lacking RI) or DR-26 (lacking RII) indicated that all three receptors were necessary for efficient down-regulation. Down-regulation experiments, utilizing TGF-β-treated 293 cells transiently expressing different combinations of these receptors indicated that neither RII or RIII were down-regulated when expressed alone and that RI was required for maximal down-regulation of RII. RII and RIII were partially down-regulated when these receptors were coexpressed in the absence of RI (in R-1B and 293 cells). Surprisingly, TGF-β receptors were partially down-regulated in Mv1Lu, A549, and 293 cells treated with TGF-β1 at 4°C. Microscopic examination of 293 cells coexpressing RI fused to green fluorescent protein (RI–GFP) and RII indicated that, after treatment with TGF-β1 at 4°C, RI–GFP formed aggregates at the cell surface at this temperature. RI–GFP was not detected at the surface of these cells after TGF-β1 treatment at 37°C. Our results suggest a two phase mechanism for TGF-β1 receptor down-regulation involving receptor modulation (aggregation) at the cell surface and internalization.     

TβRIII independently binds type I and type II TGF-β receptors to inhibit TGF-β signaling

Transforming growth factor-β (TGF-β) receptor oligomerization has important roles in signaling. Complex formation among type I and type II (TβRI and TβRII) TGF-β receptors is well characterized and is essential for signal transduction. However, studies on their interactions with the type III TGF-β coreceptor (TβRIII) in live cells and their effects on TGF-β signaling are lacking. Here we investigated the homomeric and heteromeric interactions of TβRIII with TβRI and TβRII in live cells by combining IgG-mediated patching/immobilization of a given TGF-β receptor with fluorescence recovery after photobleaching studies on the lateral diffusion of a coexpressed receptor. Our studies demonstrate that TβRIII homo-oligomerization is indirect and depends on its cytoplasmic domain interactions with scaffold proteins (mainly GIPC). We show that TβRII and TβRI bind independently to TβRIII, whereas TβRIII augments TβRI/TβRII association, suggesting that TβRI and TβRII bind to TβRIII simultaneously but not as a complex. TβRIII expression inhibited TGF-β–mediated Smad2/3 signaling in MDA-MB-231 cell lines, an effect that depended on the TβRIII cytoplasmic domain and did not require TβRIII ectodomain shedding. We propose that independent binding of TβRI and TβRII to TβRIII competes with TβRI/TβRII signaling complex formation, thus inhibiting TGF-β–mediated Smad signaling.     

Expression of nitric oxide synthase and transforming growth factor-beta in crush-injured tendon and synovium

This study examined the expression of inducible nitric oxide synthase (iNOS) and transforming growth factor-beta (TGF-beta) in macrophage infiltrates within crush-injured digital flexor tendon and synovium of control rats and rats treated with N(G)-nitro-1-arginine methyl ester (L-NAME) (5 mg/kg). Release of TGF-beta from organ cultures of tendon, muscle, and synovium, and the effects of L-NAME treatment (in vitro and in vivo), on adhesion of peritoneal macrophages to epitenon monolayers were also investigated. The results showed that during normal tendon healing the levels of TGF-beta are high at first and gradually decrease after 3 weeks of injury to slightly above control uninjured levels. However, when L-NAME was administered at the time of injury, the macrophage infiltrates were expressing high levels of TGF-beta even at 5 weeks after the injury, with no evidence of reduction. In the standard injury, iNOS activity was greatest at the acute phase of the inflammatory response and then gradually returned to normal. Treatment with L-NAME, however, resulted in inhibition of iNOS activity at 3 days and a reduction in the activity at the later time points examined after injury. We also found greatly increased levels of adhesion of peritoneal macrophages from L-NAME-treated rats to epitenon monolayers in vitro, which reflect a chronic imbalance in expression of TGF-beta, which is overexpressed, and nitric oxide, which is underexpressed. The results of the current study show that formation of nitric oxide is an important event in the course of tendon healing since its inhibition results in chronic inflammation and fibrosis due to an imbalance in TGF-beta expression in vivo.     

Enhancement of the antagonistic potency of transforming growth factor-beta receptor extracellular domains by coiled coil-induced homo- and heterodimerization

Transforming growth factor-beta (TGF-beta) plays a causal role in several human pathologies including fibrotic diseases and metastasis. TGF-beta signaling is mediated through its interaction with three types of cell surface receptors, RI, RII, and RIII. The soluble ectodomains of RII and RIII bind to TGF-beta, making them attractive candidates to sequester TGF-beta and inhibit its activity. To optimize the activity of the ectodomains, we studied the effect of artificially dimerizing them upon their kinetics of binding to TGF-beta using an optical biosensor and studied their antagonistic potencies using an in vitro signaling assay. We fused the RII ectodomain and the membrane-proximal ligand-binding domain of the RIII ectodomain to de novo designed heterodimerizing coil strands and demonstrated that the coil strands within the fusion proteins were capable of promoting the dimerization of the coil-tagged ectodomains. Our results indicate that coiled coil-induced dimerization of the ectodomains stabilized their interaction with TGF-beta as compared with the monomeric ectodomains. Also, in contrast to the monomeric ectodomains, which did not block signaling, the coiled coil-induced dimers were characterized by antagonistic potencies in the low nanomolar range.     

Curcumin differentially regulates TGF-beta1, its receptors and nitric oxide synthase during impaired wound healing

Wound healing is a highly ordered process, requiring complex and coordinated interactions involving peptide growth factors of which transforming growth factor-beta (TGF-beta) is one of the most important. Nitric oxide is also an important factor in healing and its production is regulated by inducible nitric oxide synthase (iNOS). We have earlier shown that curcumin (diferuloylmethane), a natural product obtained from the plant Curcuma longa, enhances cutaneous wound healing in normal and diabetic rats. In this study, we have investigated the effect of curcumin treatment by topical application in dexamethasone-impaired cutaneous healing in a full thickness punch wound model in rats. We assessed healing in terms of histology, morphometry, and collagenization on the fourth and seventh days post-wounding and analyzed the regulation of TGF-beta1, its receptors type I (tIrc) and type II (tIIrc) and iNOS. Curcumin significantly accelerated healing of wounds with or without dexamethasone treatment as revealed by a reduction in the wound width and gap length compared to controls. Curcumin treatment resulted in the enhanced expression of TGF-beta1 and TGF-beta tIIrc in both normal and impaired healing wounds as revealed by immunohistochemistry. Macrophages in the wound bed showed an enhanced expression of TGF-beta1 mRNA in curcumin treated wounds as evidenced by in situ hybridization. However, enhanced expression of TGF-beta tIrc by curcumin treatment observed only in dexamethasone-impaired wounds at the 7th day post-wounding. iNOS levels were increased following curcumin treatment in unimpaired wounds, but not so in the dexamethasone-impaired wounds. The study indicates an enhancement in dexamethasone impaired wound repair by topical curcumin and its differential regulatory effect on TGF-beta1, it's receptors and iNOS in this cutaneous wound-healing model.     

Ligand-dependent and -independent interactions with the transforming growth factor type II and I receptor subunits reside in the aminoterminal portion of the ectodomain of the type III subunit

Summary The type III receptor for transforming growth factor beta (TGFβ), which exhibits no kinase activity, binds TGFβ1 and TGFβ2 and is involved in assembly and activity of the multi-subunit TGFβ signal transduction complex. Recently we showed that TGFβ receptor type III (TβRIII) can participate in a complex composed of the dimeric TGFβ ligand and a type III, II, and I receptor subunit. The interaction of the TβRIII subunit with TβRII is TGFβ-dependent, whereas interaction with TβRI is TGFβ-independent. Here we use coexpression of the three types of TGFβ receptors in baculoviral-infected insect cells to determine which parts of the unglycosylated TβRIII receptor participate in the binding of TGFβ, the TGFβ-dependent interaction with TβRII and the TGFβ-independent interaction with TβRI. The results suggest that the first 500 amino acid residues in the aminoterminal portion of TβRIII exhibit all three properties.     

Influence of a single loading episode on gene expression in healing rat Achilles tendons

Mechanical loading stimulates tendon healing via mechanisms that are largely unknown. Genes will be differently regulated in loaded healing tendons, compared with unloaded, just because of the fact that healing processes have been changed. To avoid such secondary effects and study the effect of loading per se, we therefore studied the gene expression response shortly after a single loading episode in otherwise unloaded healing tendons. The Achilles tendon was transected in 30 tail-suspended rats. The animals were let down from the suspension to load their tendons on a treadmill for 30 min once, 5 days after tendon transection. Gene expression was studied by Affymetrix microarray before and 3, 12, 24, and 48 h after loading. The strongest response in gene expression was seen 3 h after loading, when 150 genes were up- or downregulated (fold change ≥2, P ≤ 0.05). Twelve hours after loading, only three genes were upregulated, whereas 38 were downregulated. Fewer than seven genes were regulated after 24 and 48 h. Genes involved in the inflammatory response were strongly regulated at 3 and 12 h after loading; this included upregulation of iNOS, PGE synthase, and IL-1β. Also genes involved in wound healing/coagulation, angiogenesis, and production of reactive oxygen species were strongly regulated by loading. Microarray results were confirmed for 16 selected genes in a repeat experiment (N = 30 rats) using real-time PCR. It was also confirmed that a single loading episode on day 5 increased the strength of the healing tendon on day 12. In conclusion, the fact that there were hardly any regulated genes 24 h after loading suggests that optimal stimulation of healing requires a mechanical loading stimulus every day.     

Biomechanical analysis of intrinsic tendon healing in vitro and the effects of vitamins A and E

This in vitro study was designed to test the hypothesis that flexor tendons have an intrinsic capability to heal as judged by rigorously defined biomechanical criteria. Rabbit forepaw flexor tendons (n = 114) were transected and repaired in zone II and cultured in standard media or media supplemented with vitamin A or E. Tenorrhaphies were disrupted at 0, 2, 8, and 12 weeks after repair by fixed-speed tensiometry. The following parameters were calculated: true stress, true strain, and normalized energy absorption. Statistical analysis was by Student's t test. Peak stress (strength) increased equally in all groups over time (p less than 0.05), while normalized energy absorption (toughness) was markedly higher in the vitamin E-treated group after 2 weeks' incubation (p less than 0.05). These data represent the first biomechanical evidence of intrinsic tendon healing in vitro. Biomechanical methodology and vitamin effects are discussed in detail.     

Expression of a functional high-affinity IgG receptor, Fc gamma RI, on human mast cells: Up-regulation by IFN-gamma

Biologically relevant activation of human mast cells through Fc receptors is believed to occur primarily through the high-affinity IgE receptor Fc epsilon RI. However, the demonstration in animal models that allergic reactions do not necessarily require Ag-specific IgE, nor the presence of a functional IgE receptor, and the clinical occurrence of some allergic reactions in situations where Ag-specific IgE appears to be lacking, led us to examine the hypothesis that human mast cells might express the high-affinity IgG receptor Fc gamma RI and in turn be activated through aggregation of this receptor. We thus first determined by RT-PCR that resting human mast cells exhibit minimal message for Fc gamma RI. We next found that IFN-gamma up-regulated the expression of Fc gamma RI. This was confirmed by flow cytometry, where Fc gamma RI expression on human mast cells was increased from approximately 2 to 44% by IFN-gamma exposure. Fc epsilon RI, Fc gamma RII, and Fc gamma RIII expression was not affected. Scatchard plots were consisted with these data where the average binding sites for monomeric IgG1 (Ka = 4-5 x 108 M-1) increased from approximately 2,400 to 12,100-17,300 per cell. Aggregation of Fc gamma RI on human mast cells, and only after IFN-gamma exposure, led to significant degranulation as evidenced by histamine release (24.5 +/- 4.4%): and up-regulation of mRNA expression for specific cytokines including TNF-alpha, GM-CSF, IL-3 and IL-13. These findings thus suggest another mechanism by which human mast cells may be recruited into the inflammatory processes associated with some immunologic and infectious diseases.     

Predominant intracellular localization of the type I transforming growth factor-beta receptor and increased nuclear accumulation after growth arrest

Transforming growth factor-beta (TGF-beta) signaling requires the functional interaction of two distinct receptors, type I (RI) and type II (RII), at the cell surface. Exposure of cells to TGF-beta results in receptor internalization and down-regulation (Zwaagstra et al., 1999, Exp. Cell Res. 252, 352362); however, little is known about the subsequent fate of RI or RII. In this study the cellular distribution of RI was examined in cells before and after treatment with ligand. RI was localized by immunocytochemistry and confocal microscopy using two polyclonal antisera directed against two different epitopes, one in the C-terminal region and one in the N-terminal region of the cytoplasmic domain. The majority of RI molecules in untreated MvlLu and A549 cells were found to be intracellular. Treatment of MvlLu and A549 cells with 100 pM TGF-beta1 for 24 h at 37 degrees C caused a redistribution of surface RI on MvlLu cells, as evidenced by surface RI aggregation. Unexpectedly, this TGF-beta1 treatment also caused redistribution and accumulation of intracellular RI in and around the nucleus for both MvlLu and A549 cells. Nuclear accumulation of RI was also promoted independently of ligand receptor activation by treatment of MvlLu cells with olomoucine, an agent that results in growth arrest. The capacity of RI to localize in the nucleus was confirmed by microscopic examination of 293 cells transiently expressing RI fused to green fluorescent protein (RI-GFP). Olomoucine treatment of these cells resulted in the movement of RI-GFP into the nucleus. Our results indicate that growth arrest alters intracellular transport/routing of RI and may indicate that RI functions not only at the cell surface but inside the cell as well.