Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation.

Solid tumor growth triggers a wound healing response. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts (also referred to as cancer-associated fibroblasts) primarily, but not exclusively, in response to transforming growth factor-β (TGF-β). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among proteases implicated in stroma remodeling, matrix metalloproteinases (MMPs), including MMP-9, play a prominent role. Recent evidence indicates that MMP-9 recruitment to the tumor cell surface enhances tumor growth and invasion. In the present work, we addressed the potential relevance of MMP-9 recruitment to and activity at the surface of fibroblasts. We show that recruitment of MMP-9 to the fibroblast cell surface occurs through its fibronectin-like (FN) domain and that the molecule responsible for the recruitment is lysyl hydroxylase 3 (LH3). Functional assays suggest that both pro- and active MMP-9 trigger α-smooth muscle actin expression in cultured fibroblasts, reflecting myofibroblast differentiation, possibly as a result of TGF-β activation. Moreover, the recombinant FN domain inhibited both MMP-9-induced TGF-β activation and α-smooth muscle actin expression by displacing MMP-9 from the fibroblast cell surface. Together our results uncover LH3 as a new docking receptor of MMP-9 on the fibroblast cell surface and demonstrate that the MMP-9 FN domain is essential for the interaction. They also show that the recombinant FN domain inhibits MMP-9-induced TGF-β activation and fibroblast differentiation, providing a potentially attractive therapeutic reagent toward attenuating tumor progression where MMP-9 activity is strongly implicated.

Beta2-adrenergic receptor agonists reduce proliferation but not protein synthesis of periodontal fibroblasts stimulated with platelet-derived growth factor-BB

OBJECTIVE Catecholamines released from β-adrenergic neurons upon stress can interfere with periodontal regeneration. The cellular mechanisms, however, are unclear. Here, we assessed the effect of catecholamines on proliferation of periodontal fibroblasts. METHODS Fibroblasts from the gingiva and the periodontal ligament were exposed to agonists of the β-adrenergic receptors; isoproterenol (ISO, non-selective β-adrenergic agonist), salbutamol (SAL, selective β2-adrenergic receptor agonist) and BRL 37344 (BRL selective β3-receptor agonist). Proliferation was stimulated with platelet-derived growth factor-BB (PDGF-BB). Pharmacological inhibitors and gene expression analysis further revealed β-adrenergic signalling. RESULTS Gingiva and periodontal ligament fibroblast express the β2-adrenergic receptor. ISO and SAL but not BRL decreased proliferation of fibroblasts in the presence of PDGF-BB. The inhibitory effect of β-adrenergic signalling on proliferation but not protein synthesis in response to PDGF-BB was reduced by propranolol, a non-selective β-adrenergic antagonist. CONCLUSIONS These results suggest that β2-receptor agonists can reduce the mitogenic response of periodontal fibroblasts. These data add to the compelling concept that blocking of β2-receptor signalling can support tissue maintenance and regeneration.

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae

Messenger RNAs coding for growth factors and receptor tyrosine kinases were measured by quantitative competitive and by semi-quantitative reverse-transcription polymerase chain reaction in whole and dissected chick inner ears. The fibroblast growth factor (FGF) receptor 1 chick embryonic kinase (CEK) 1 was expressed in all structures examined (otocyst, hatchling whole cochlea, cochlear nerve ganglion, and cochlear and vestibular sensory epithelia), although slightly more heavily in the otocyst. The related fibroblast growth factor receptors CEK 2 and 3 were preferentially expressed in the nerve ganglion and in the vestibular sensory epithelium, respectively. FGF 1 mRNA was low in early development, increasing to mature levels at around embryonic age 11 days, while FGF2, mRNA was expressed at constant levels at all ages. In response to ototoxic damage, FGF1 mRNA levels were increased in the early damaged cochlear sensory epithelium. Immunohistochemistry for CEK1 showed that normal hair cells expressed the receptor heavily on the hair cell stereocilia, while with early damage, CEK1 came to be expressed heavily on the apical surfaces of the supporting cells. In normal chicks, the CEK4 and CEK8 eph-class receptor tyrosine kinases were expressed relatively heavily by the cochlear nerve ganglion, and CEK10 was expressed relatively heavily by the cochlear hair cell sensory epithelium. The results suggest that the FGF system may be involved in the response of the cochlear epithelium to ototoxic damage. The eph-class receptor tyrosine kinase CEK10 may be involved in cell interactions in the cochlear sensory epithelium, while CEK4 and CEK8 may play a role in the cochlear innervation.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in

Expression of insulin-like growth factor-II and its receptor in pediatric and adult adrenocortical tumors

Background: Adrenocortical tumors are heterogeneous neoplasms with incompletely understood pathogenesis. IGF-II overexpression has been consistently demonstrated in adult adrenocortical carcinomas. Objectives: The objective of the study was to analyze expression of IGF-II and its receptor (IGF-IR) in pediatric and adult adrenocortical tumors and the effects of a selective IGF-IR kinase inhibitor (NVP-AEW541) on adrenocortical tumor cells. Patients: Fifty-seven adrenocortical tumors (37 adenomas and 20 carcinomas) from 23 children and 34 adults were studied. Methods: Gene expression was determined by quantitative real-time PCR. Cell proliferation and apoptosis were analyzed in NCI H295 cells and a new cell line established from a pediatric adrenocortical adenoma. Results: IGF-II transcripts were overexpressed in both pediatric adrenocortical carcinomas and adenomas. Otherwise, IGF-II was mainly overexpressed in adult adrenocortical carcinomas (270.5 +/- 130.2 vs. 16.1 +/- 13.3; P = 0.0001). IGF-IR expression was significantly higher in pediatric adrenocortical carcinomas than adenomas (9.1 +/- 3.1 vs. 2.6 +/- 0.3; P = 0.0001), whereas its expression was similar in adult adrenocortical carcinomas and adenomas. IGF-IR expression was a predictor of metastases in pediatric adrenocortical tumors in univariate analysis (hazard ratio 1.84; 95% confidence interval 1.28 -2.66; P = 0.01). Furthermore, NVP-AEW541 blocked cell proliferation in a dose-and time-dependent manner in both cell lines through a significant increase of apoptosis. Conclusion: IGF-IR overexpression was a biomarker of pediatric adrenocortical carcinomas. Additionally, a selective IGF-IR kinase inhibitor had antitumor effects in adult and pediatric adrenocortical tumor cell lines, suggesting that IGF-IR inhibitors represent a promising therapy for human adrenocortical carcinoma.

Non-expression of insulin-like growth factor-I receptor is associated with apoptosis: an ultrastructural study on rat ameloblasts

Insulin-like growth factor-I (IGF-I) is a preiotrophic polypeptide which appears to have roles both as a circulating endocrine hormone and as a locally synthesized paracrine or autocrine tissue factor. IGF-I plays a major role in regulating the growth of cells in vivo and in vitro and initiates metabolic and mitogenic processes in a wide variety of cell types by binding to specific type I receptors in the plasma membrane, In this study, we report the distribution of IGF-I receptors in odontogenic cells at the ultrastructural level using the high resolution protein A-gold technique, In the pre-secretory stage, very little gold label was visible over the ameloblasts and odontoblasts, During the secretory stage the label was mostly seen in association with the cell membranes and endoplasmic reticulum of the ameloblasts. Lysosome-like elements in the post-secretory stage were labelled as well as multivesicular dense bodies, Very little labelling was encountered in the ameloblasts in the transitional stage, where apoptotic bodies were clearly visible, The maturation stage also exhibited labelling of the secretory-like granules in the distal surface. The presence of gold particles over the plasma membrane is an indication that IGF-I receptor is a membrane-bound receptor. Furthermore, the intracellular distribution of the label over the endoplasmic reticulum supports the local synthesis of the IGF-I receptor. The absence of labelling over the transitional ameloblasts suggests that the transitional stage may require the non-expression of IGF-I as a prerequiste or even a trigger for apoptosis.

Vascular density and distribution of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) are significantly higher in patients with deeply infiltrating endometriosis affecting the rectum

Objective: To analyze vascular density and immunolocalization of angiogenic vascular endothelial growth factor (VEGF) and its receptor Flk-1 in the proliferative and secretory eutopic human endometrium. and in three different sites of endometriosis: the ovary, bladder, and rectum. Design: Prospective study. Setting: University hospital. Patient(s): Thirty women with endometriosis (10 ovarian, 1.0 bladder, 10 rectal) and 32 control women (10 proliferative endometrium, 10 secretory endometrium, 4 normal ovary, 4 normal bladder, 4 normal rectum). Intervention(s): Normal endometrial samples were obtained from women during laparoscopic ablation of subserous myoma, and biopsy specimens of endometriosis were obtained from patients undergoing surgery for the diagnosis and treatment of endometriosis. Normal tissues of ovary, bladder, and rectum were obtained from these organs beside the lesions of endometriosis. Main Outcome Measure(S): Blood vessels were quantified according to the number of von Willebrand factor-positive endothelial cells. The VEGF and Flk-1 distribution were evaluated semiquantitatively by immunohistochemical staining. Result(s): More blood vessels were found in cases of endometriosis, particularly rectal endometriosis, compared with the respective control samples and with the eutopic endometrium, and they were localized in endometrial stroma around the glands. The VEGF and Flk-1 expression levels were also higher in cases of endometriosis, especially rectal endometriosis. Conclusion(s): Vascularization and VEGF and Flk-1 expression are significantly higher in deeply infiltrating endometriosis affecting the rectum, reinforcing the hypothesis that antiangiogenesis therapy may constitute a new modality of treatment, especially in cases of deep endometriosis involving the rectum.

NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear H-1-N-15 NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta -strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. N-15 longitudinal and transverse relaxation rates, and {H-1}-N-15 heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85 +/- 0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides. (C) 2001 Academic Press.

Growth hormone receptor and insulin-like growth factor-I immunoreactivity in osteoclast-like cells during tooth eruption in the toothless (Osteopetrotic) rat following treatment with colony-stimulating factor-1

In the toothless (tl/tl) osteopetrotic rat, teeth form but fail to erupt. Treatment of tl/tl rats with colony-stimulating factor-1 (CSF-1) activates bone resorption by osteoclasts, permits tooth eruption, and up-regulates the immunoreactivity of bone marrow mononuclear cells to growth hormone receptor (GHr) and insulin-like growth factor (IGF)-I. This study examined the distribution of tartrate-resistant acid phosphatase (TRAP) and immunoreactivity for GHr and IGF-I in osteoclast-like cells located on the alveolar bone margin, adjacent to the lower first molar crown, in 14-day-old normal and tl/tl rats, following treatment with CSF-1. Osteoclast-like cells demonstrated a positive reaction for TRAP, GHr, and IGF-I in all groups. However, in tl/tl tissue, osteoclast-like cells were generally negative for GHr. There was no significant difference in the total number of TRAP, GHr, and IGF-I-positive osteoclast-like cells on the adjacent bone margin in normal, normal treated with CSF-1, and tl/tl rats. CSF-1 treatment of the tl/tl rat significantly increased the total number of osteoclast-like cells, which were positive for TRAP (p < 0.001), GHr (p < 0.05) and IGF-I (P < 0.01).

Genetic- or transforming growth factor-beta 1-induced changes in epidermal peroxisome proliferator-activated receptor beta/delta expression dictate wound repair kinetics.

Advances in wound care are of great importance in clinical injury management. In this respect, the nuclear receptor peroxisome proliferator-activated receptor (PPAR)beta/delta occupies a unique position at the intersection of diverse inflammatory or anti-inflammatory signals that influence wound repair. This study shows how changes in PPARbeta/delta expression have a profound effect on wound healing. Using two different in vivo models based on topical application of recombinant transforming growth factor (TGF)-beta1 and ablation of the Smad3 gene, we show that prolonged expression and activity of PPARbeta/delta accelerate wound closure. The results reveal a dual role of TGF-beta1 as a chemoattractant of inflammatory cells and repressor of inflammation-induced PPARbeta/delta expression. Also, they provide insight into the so far reported paradoxical effects of the application of exogenous TGF-beta1 at wound sites.

An SH2 domain-containing 5' inositolphosphatase inhibits insulin-induced GLUT4 translocation and growth factor-induced actin filament rearrangement.

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and growth factor signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- 21% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like growth factor I (IGF-I)-, and platelet-derived growth factor-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat fibroblast cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, growth factor-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly growth factor-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, growth factor-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.

Insulin-like growth factor and growth hormone receptor in postpartum lactating beef cows

The objective of this study was to evaluate the plasma concentrations of insulin-like growth factor-I (IGF-I), and the mRNA hepatic expression of IGF-I and of the growth hormone receptors GHR and GHR 1A, in postpartum beef cows. Four Angus and four crossbred (Angus x Nelore) postpartum suckled beef cows were used. Liver and blood samples were collected every 10 days, from calving to 40 days postpartum, for gene expression and for β-hydroxybutyrate and IGF-I assays, respectively. Samples for progesterone assay were collected every other day, from day 10 to 40 postpartum. Three cows ovulated before 40 days postpartum. IGF-I concentration was higher in Angus x Nelore than in Angus cows. There was no difference in the expression of GHR, GHR 1A and IGF-I according to breed or ovulatory status. IGF-I concentrations were higher in crossbred cows, but have not changed according to postpartum ovulatory status. Moreover, changes in postpartum IGF-I concentrations are not associated with changes in liver GHR, GHR 1A and IGF-I mRNA expression in either breed.