Novel Peptides Derived from Sea Cucumber Intestine Promotes Osteogenesis by Upregulating Integrin-Mediated Transdifferentiation of Growth Plate Chondrocytes to Osteoblasts.

The sea cucumber intestine is a major by-product of sea cucumber processing and contains high levels of protein. In this study, we isolated and identified 28 novel osteogenic peptides from sea cucumber intestinal hydrolysis by the activity-tracking method for the first time. In vitro experimental results showed that compared with high molecular weight, the peptides from sea cucumber intestine (SCIP) with molecular weight <3 kDa more significantly promoted the proliferation and mineralized nodules of MC3T3-E1 cell and exhibited potential integrin binding capacity. In vivo experimental results showed that the SCIP supplement significantly increased the longitudinal bone length and elevated the height of the growth plate (especially the hypertrophic zone, 37.2%, p < 0.01) in adolescent mice. Further, immunofluorescence labeling results indicated that the SCIP supplement increased chondrocyte transdifferentiate to osteoblast in the growth plate close to the diaphysis. Mechanistically, transcriptome analysis revealed that the SCIP supplement induced the dedifferentiation of chondrocyte to osteoprogenitor cell via integrin-mediated histone acetylation and then redifferentiated to osteoblast via integrin-mediated Wnt/ß-catenin signaling. These results reported for the first time that sea cucumber intestine had the potential to develop into a dietary supplement for promoting osteogenic, and provide new evidence for the mechanism of dietary promotes chondrocyte to osteoblast transdifferentiation.

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications.

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.

Pectasol-C Modified Citrus Pectin targets Galectin-3-induced STAT3 activation and synergize paclitaxel cytotoxic effect on ovarian cancer spheroids.

Here we sought to determine the relationship between STAT3 activity and Galectin-3 (Gal-3) and to investigate the cytotoxic effect of PectaSol-C Modified Citrus Pectin (Pect-MCP) as a specific competitive inhibitor of Galectin-3 (Gal-3) in combination with Paclitaxel (PTX) to kill the ovarian cancer cell SKOV-3 multicellular tumor spheroid (MCTS). To this order, SKOV-3 cells in 2D and 3D cultures were treated with exogenous Gal-3 for the assessment of STAT3 activity. Two-way ANOVA main effect and IC50 of each drug Paclitaxel (PTX) and Pect-MCP or in combination were obtained from MTT assay results. The phosphorylated STAT3 levels, migration, invasion, integrin mRNA and p-AKTser473 levels were assessed in the absence or presence of each drug alone or in combination. Gal-3 expression levels were assessed in human serous ovarian cancer (SOC) specimens and its correlation with different integrin mRNA levels was further assessed. Our results showed that Gal-3 expression level was significantly increased in MCTS compared to monolayer SKOV-3 cells which triggered STAT3 phosphorylation. Moreover, Pect-MCP synergized with PTX to kill SKOV3 MCTS through abrogation of STAT3 activity and reduced expression of its downstream target HIF-1α, reduced integrin mRNA levels, and subsequently decreased AKT activity. There were higher expression levels of Gal-3 in human high-grade SOC specimens compared to the normal ovary and borderline SOC which positively and significantly correlated with α5, ß2 and ß6 integrin mRNA levels. Together, these results revealed for the first time that Pect-MCP could be considered as a potential drug to enhance the PTX effect on ovarian cancer cells MCTS through inhibition of STAT3 activity.

Roles of integrin in tumor development and the target inhibitors.

Integrin is a large family of cell adhesion molecules (CAMs) which involves in the interaction of cells/cells and cells/ extracellular matrix (ECM) to mediate cell proliferation, differentiation, adhesion, migration, etc. In recent years, aberrant expression of integrin has been clearly found in many tumor studies, indicating that integrin is closely related to tumor formation and development. Meanwhile, it has effects on tumor cell differentiation, cell migration, proliferation and tumor neovascularization. The study of drugs targeting integrins is of great significance for the clinical treatment of tumors. Because of its important role in tumorigenesis and development, integrin has become a promising target for the treatment of cancer. This review summarizes the role of integrin in tumor development and the current state of integrin inhibitors to provide a valuable reference for subsequent research.

[Effect of celastrol in inhibiting metastasis of lung cancer cells by influencing Akt signaling pathway and expressing integrins].

Celastrol is a type of quinone methyl triterpene isolated from traditional Chinese medicine Tripterygium wilfordii, with pharmacological activities, like anti-inflammatory, immunosuppression and anti-tumor. This study focused on the effects of celastrol on adhesion, migration and invasion of lung cancer cells. The migration inhibition of lung cancer cells induced by celastrol was detected by the scratch test. The invasion inhibition of lung cancer cells induced by celastrol was measured by the transwell experiment. RT-PCR and Western blot were used to determine the effect of different concentrations of celastrol in integrin family and Akt signaling pathway in lung cancer cells. The results showed that celastrol inhibited adhesion, migration and invasion of lung cancer cells and expressions of integrins ß3, ß4, αv and phosphorylated Akt, GSK-3ß, c-Raf, PDK1 in Akt signal pathway in a dose-dependent manner. Therefore, the study implies that Celastrol could inhibit the metastasis of lung cancer cells by suppressing Akt signaling pathway and expression of integrins.

[Study on effect of naringenin in inhibiting migration and invasion of breast cancer cells and its molecular mechanism].

OBJECTIVE: To study the effect of nadroparin in the migration of breast cancer cells MDA-MB-231 and its action mechanism. METHOD: The MTT test was adopted to observe the effect of different concentrations of naringenin on the growth capacity of breast cancer cells MDA-MB-231. Wound healing and transwell experiment analysis were conducted to detect the effect of naringenin on the migration of breast cancer cells MDA-MB-231. Western blotting was adopted to investigate the effect of naringenin on protein expressions of MDA-MB-231 cell Integrin ß3, ß1 and matrix metalloproteinase MMP-2 and MMP-9. The computer virtual docking technique was used to evaluate the combining capacity of naringenin and Integrin ß3 in vitro. RESULT: Naringenin inhibited the migration of MDA-MB-231 cells in a dose-dependent manner. In wound healing and transwell experiments, with the increase in the concentration of naringenin, the number of migrant MDA-MB-231 cells and the invasion capacity of breast cancer cells decreased. Naringenin could inhibit the protein expression of Integrin ß3 in a dose-dependent manner, but with unobvious effect on expression of Integrin ß1. Besides, naringenin could significantly inhibit the protein expressions of MMP-2 and MMP-9. The results of the computer virtual docking showed a negative value in the combining capacity between naringenin and Integrin ß3, indicating the high affinity between them. CONCLUSION: Naringenin can inhibit the growth capacity of breast cancer cells MDA-MB-231 and block the migration and invasion of breast cancer cells MDA-MB-231. Its mechanism is to down-regulate MMP-2 and MMP-9 expressions after combining with Integrin ß3.

Protopine inhibits heterotypic cell adhesion in MDA-MB-231 cells through down-regulation of multi-adhesive factors.

BACKGROUND: A Chinese herb Corydalis yanhusuo W.T. Wang that showed anticancer and anti-angiogenesis effects in our previous studies was presented for further studies. In the present study, we studied the anticancer proliferation and adhesion effects of five alkaloids which were isolated from Corydalis yanhusuo. MATERIALS AND METHODS: MTT dose response curves, cell migration assay, cell invasion assay, as well as three types of cell adhesive assay were performed on MDA-MB-231 human breast cancer cells. The mechanism of the compounds on inhibiting heterotypic cell adhesion were further explored by determining the expression of epidermal growth factor receptor (EGFR), Intercellular adhesion molecule 1 (ICAM-1), αv-integrin, ß1-integrin and ß5-integrin by western blotting assay. RESULTS: In five tested alkaloids, only protopine exhibited anti-adhesive and anti-invasion effects in MDA-MB-231 cells, which contributed to the anti-metastasis effect of Corydalis yanhusuo. The results showed that after treatment with protopine for 90 min, the expression of EGFR, ICAM-1, αv-integrin, ß1-integrin and ß5-integrin were remarkably reduced. CONCLUSION: The present results suggest that protopine seems to inhibit the heterotypic cell adhesion between MDA-MB-231 cells, and human umbilical vein endothelial cells by changing the expression of adhesive factors.

Leaders in cell adhesion: an interview with Richard Hynes, pioneer of cell-matrix interactions. Interview by Pamela Cowin.

On a recent visit Richard O Hynes, FRS, HHMI, Daniel K. Ludwig Professor for Cancer Research at the Koch Institute for Integrative Cancer Research, MIT, graciously agreed to be interviewed in person for the first in Cell Communication and Adhesion's series on "Leaders in Cell Adhesion". In this interview we discussed three things: 1) the early role of family, mentors, and luck on his career path; 2) his major discoveries of fibronectin, integrins and the evolution of extracellular matrix proteins; and 3) his role in, and thoughts on, current science policy. This interview reveals his characteristic calmness and infectious optimism, his spontaneous and down to earth sense of humor, and his great ability to place scientific questions in perspective. The interview, carried out on April 30(th) 2013 is reported here verbatim with only minor editing for clarity.

Complementary roles of retinoic acid and TGF-ß1 in coordinated expression of mucosal integrins by T cells.

α(4) and ß(7) integrins, such as α(4)ß(1), α(4)ß(7), and α(E)ß(7), are major integrins required for migration of leukocytes into mucosal tissues. The mechanisms responsible for coordinated expression of these three integrins have been poorly elucidated to date. We report that expression of the Itg-α(4) subunit by both CD4(+) and CD8(+) T cells requires the retinoic acid signal. In contrast, transcription of Itg-α(E) genes is induced by the transforming growth factor-ß1 (TGFß1) signal. Expression of Itg-ß(7) is constitutive but can be further increased by TGFß1. Consistently, expression of α(4)-containing integrins is severely suppressed in vitamin A deficiency with a compensatory increase of α(E)ß(7), whereas expression of Itg-α(E) and Itg-ß(7) is decreased in TGFß-signal deficiency with a compensatory increase in α(4)ß(1). The retinoic acid-mediated regulation of α(4) integrins is required for specific migration of T cells in vitro and in vivo. These results provide central regulatory mechanisms for coordinated expression of the major mucosal integrins.

Integrin and talin in the jellyfish Podocoryne carnea.

We have isolated an integrin-beta and -alpha subunit from Podocoryne carnea (Cnidaria, Hydrozoa) and studied their expression in the life-cycle and during cell migration, in vitro transdifferentiation and regeneration. Comparison of the integrin expression pattern with a Podocoryne talin homologue by RT-PCR demonstrates that all three genes are maternal messages and continuously expressed in the life-cycle, in medusa development and in all medusae tissues. In situ hybridisation experiments confirm co-expression of both integrin subunits in the different life-stages. Integrin expression was furthermore studied in isolated striated muscle induced to transdifferentiate to new cell types, or grafted on ECM where the muscle adheres and migrates. Integrin expression was maintained continuously throughout both processes. These results suggest that in Podocoryne carnea processes such as cell migration and differentiation are not controlled by up- or downregulation of alternative integrin subunits, but by a single integrin heterodimer which activates different downstream signalling cascades.

Roles for beta(pat-3) integrins in development and function of Caenorhabditis elegans muscles and gonads.

Heterodimeric integrin receptors for extracellular matrix (ECM) play vital roles in bidirectional signaling during tissue development, organization, remodeling, and repair. The beta integrin subunit cytoplasmic domain is essential for transmission of many of these signals and overexpression of an unpaired beta tail in cultured cells inhibits endogenous integrins. Unlike vertebrates, which have at least nine beta subunit genes, the nematode Caenorhabditis elegans expresses only one beta subunit (betapat-3), and a null mutation in this gene causes embryonic lethality. To determine the functions of integrins during larval development and in adult tissues, we have taken a dominant negative approach by expression of an HA-betatail transgene composed of a hemagglutinin (HA) epitope tag extracellular domain connected to the betapat-3 transmembrane and cytoplasmic domains. Expression of this transgene in muscle and gonad, major sites of integrin expression, caused a variety of phenotypes dependent on the level of transgene expression. Abnormalities in body wall and sex muscles led to uncoordinated movement and egg-laying defects. Significant anomalies in migration and pathfinding were caused by tissue-specific expression of HA-betatail in the distal tip cells (DTC), the cells that direct gonad morphogenesis. A pat-3 gene with Tyr to Phe mutations in the cytoplasmic domain was able to rescue pat-3 null animals but also showed DTC migration defects. These results show that betapat-3 plays important roles in post-embryonic organogenesis and tissue function.

Effects of deregulated Raf activation on integrin, cytokine-receptor expression and the induction of apoptosis in hematopoietic cells.

The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines. The conditionally active Raf proteins were regulated by beta-estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (deltaRaf:ER). Continuous deltaRaf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/deltaRaf:ER cells as they grew in large aggregated masses (>100 cells) whereas the parental cytokine-dependent FDC-P1 cells grew in smaller grape-like clusters (< 10 cells). FD/deltaRaf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and beta-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of deltaRaf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited 3H-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation.

Up-regulation of alpha 2 beta 1 integrin cell-surface expression protects A431 cells from epidermal growth factor-induced apoptosis.

High epidermal growth factor (EGF) concentration (10(-8) M) induces inhibition of A431 cell proliferation, resulting in part from an apoptotic process. For some cells escaping this process, proliferation was associated with a decrease in apoptosis. Moreover, these surviving cells displayed marked morphological changes consisting of filopodia formation and cell aggregation. Disrupting cell-cell contacts by lowering extracellular calcium concentration reversed the resistance process, suggesting that apoptosis protection by aggregation may involve intercellular adhesion and cell-cell survival signals probably mediated by calcium-requiring molecules such as integrins. From a panel of integrins tested, only alpha 2 beta 1 integrin cell-surface expression was up-regulated after high apoptotic EGF treatment, and this up-regulation was not observed under a growth-stimulatory EGF concentration (10(-11) M). Double-labeling analysis (alpha 2 beta 1/DNA) implicated alpha 2 beta 1 integrin in the resistance process since 99% of cells that up-regulated alpha 2 beta 1 integrin survived a high dose of EGF. Moreover, the involvement of alpha 2 beta 1 integrin up-regulation in the survival of A431 cells that escape EGF-induced apoptosis was verified using the blocking anti-alpha 2 beta 1 integrin antibody, which was shown to decrease the survival of EGF-stimulated cells. Furthermore, under our culture conditions, alpha 2 beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions, suggesting that alpha 2 beta 1 integrin is involved more directly in cell-cell interaction than in cell-substrate adhesion. Our results provide evidence that EGF-induced up-regulation of alpha 2 beta 1 integrin contributes to the enhancement of cell-cell adhesion, leading to cell aggregate formation, which permits the escape of A431 cells to EGF-induced death by alpha 2 beta 1 integrin signaling.

In vitro investigation of factors important for the delivery of an integrin-targeted nonviral DNA vector in organ transplantation.

BACKGROUND: Polylysine-molossin is a 31 amino acid synthetic peptide that has previously been demonstrated to function as a DNA vector in vitro for cell lines and for the cornea. It incorporates the 15 amino acid integrin-binding domain of the venom of the American pit viper, Crotalus molossus molossus as the targeting moiety and a chain of 16 lysines as the DNA-binding moiety. The objective of this study was to evaluate several parameters of importance for in vivo applications. METHODS: Binding and tissue distribution of the vector/DNA complexes were followed by a monoclonal antibody to the vector, or by the use of fluorescein-labeled DNA. Standard in vitro transfections were used to monitor effective gene transfer. RESULTS: (1) Optimal DNA/vector concentration. Saturation of vector/DNA binding sites on the ECV304 cell line occurred at 6 microg/ml of DNA. The concentration of vector/DNA complexes required for optimal gene transfection was found to be 2-8 microg/ml of DNA, corresponding to the concentration needed for saturation binding. (2) Optimal target cell exposure time. Vector/ DNA complexes saturated target cell binding sites within 5 min of incubation. However, lengthy exposure times (>2-3 hr) to the transfection medium were essential for substantial gene transfer. This was a consequence of two complementary factors. First, it was important that target cells be exposed to vector/DNA complexes for approximately 1 hr at 37 degrees C. Saturation of target sites at 4 degrees C and then removal of the transfection medium was much less effective. Second, exposure to chloroquine for 8-10 hr after uptake of vector/DNA complexes was essential for optimal gene transfer. (3) Inhibitory effects of serum. Exposure of complexes to even 1% serum before transfection, markedly inhibited gene transfer. However, target cells previously saturated with vector/DNA complexes and then exposed to 10% serum showed substantial gene transfer. (4) Extravasation and binding stability in vivo. Cold ex vivo perfusion of rat hearts with vector/DNA complexes demonstrated that little, if any, complex moved out of the vascular system. After transplantation of the heart, most of the complex bound to the vasculature was lost within 30 min of reestablishing the blood circulation. CONCLUSIONS: Careful attention to several parameters of little importance in vitro need to be paid for optimal in vivo application of DNA vector systems.

Role of the alpha1 and alpha2 integrin cytoplasmic domains in cell morphology, motility and responsiveness to stimulation by the protein kinase C pathway.

The alpha1beta1 and alpha2beta1 integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the alpha subunits. We expressed either full-length alpha1 integrin subunit cDNA (X1C1), full-length alpha2 integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of alpha2 subunit and the cytoplasmic domain of alpha1 (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of alpha1 subunit and the cytoplasmic domain of alpha2 (X1C2), alpha1 cDNA truncated after the GFFKR sequence (X1C0) or alpha2 cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the alpha1 and alpha2 subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the alpha1 or alpha2 cytoplasmic tail. Spreading was also influenced by the presence of an alpha subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the alpha2 cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, alpha2-cytoplasmic tail-dependent migration.

A new approach to antiplatelet therapy: inhibitor of GPIb/V/IX-vWF interaction.

Evidence has been presented that the interaction between von Willebrand factor (vWF) and its platelet membrane receptor, the GPIb/V/IX complex, plays an important role in the pathogenesis of arterial thrombosis. A monoclonal antibody against the A1 domain of vWF has been shown to inhibit thrombus formation in the animal model of arterial thrombosis. Based upon these findings, a new approach to treating arterial thrombosis has been proposed by intervening in the interaction between vWF and platelet.

The integrin alphavbeta5 is expressed on avian osteoclast precursors and regulated by retinoic acid.

Osteoclasts arise by proliferation, differentiation, and subsequent fusion of marrow-derived precursors, all processes requiring attachment to matrix. Integrins are important mediators of cell-matrix recognition and bone is rich in proteins containing the Arg-Gly-Asp motif, recognized primarily by alphav integrins. Thus, we determined if avian osteoclast precursors express integrins capable of mediating initial attachment to matrix proteins. Early, marrow-derived osteoclast precursors, when first isolated, contain no detectable alphavbeta3, but express an alphav integrin with an 80 kDa associated beta subunit. Immunoprecipitation with an antibody raised against the conserved beta5 cytoplasmic tail sequence indicates the the alphav associated the integrin is alphavbeta5. Retinoic acid is a resorptive steroid, and its exposure to early osteoclast precursors prompts a time- and dose-dependent decrease in alphavbeta5 expression, while simultaneously stimulating alphavbeta3 expression. Northern analysis reveals that retinoic acid decreases beta5 steady-state mRNA, nontranscriptionally, without altering that of alphav. The finding alphavbeta5 expression decreases under the influence of retinoic acid, an osteoclastogenic steroid, while those of alphavbeta3 rise, suggests that these closely related integrins play separate and complementary roles during osteoclast differentiation.

Phenotypic characterization of rat hepatoma cell lines and lineage-specific regulation of gene expression by differentiation agents.

Hepatoma cell lines can be characterized by their expression of hepatocyte- and biliary-specific genes and by their response to differentiating agents in a lineage-dependent manner. These characteristics can be used to map the maturational lineage position of the cell lines. Tissue-specific gene expression and regulation by heparin, dimethylsulfoxide (DMSO), and sodium butyrate (SB) were examined in three rat hepatoma cell lines and two rat liver epithelial cell lines. Based on antigenic profiles and gene expression in serum-supplemented medium, the hepatoma cell lines could be organized in distinct categories of hepatic differentiation. All three hepatomas expressed the following five genes: gamma-glutamyl transpeptidase (GGT), glutathione-S-transferase pi (Yp), glutamine synthetase, and alpha 5 and beta 1 integrin. Cell line H4AzC2 also expressed alpha-fetoprotein (AFP), albumin. IGF II receptor, and the biliary/oval cell antigens OC.2 and OC.3, a phenotype characteristic of fetal hepatocytes. FTO-2B cells lacked AFP, OC.2, and OC.3 but expressed albumin and IGF II receptor in addition to the five commonly expressed genes, consistent with a more hepatocyte-like phenotype. Cell line H5D-7 expressed neither albumin nor the IGF II receptor, but did express OC.2, OC.3, and alpha 3 integrin in addition to the five commonly expressed genes, characteristic of biliary epithelial cells. Regulation of gene expression by heparin, DMSO, and SB was examined in cells cultured in hormonally defined medium. The patterns of regulation of AFP, albumin, GGT, and Yp were dependent upon the state of differentiation of the cell. FTO-2B cells regulated genes in a manner similar to that of E16 fetal hepatocytes, H4AzC2 regulated genes characteristic of both hepatocytic and biliary lineages, and H5D.7 regulated only biliary genes. Suppression of GGT by DMSO was uniformly observed. The three cell lines expressed equal amounts of HNF-4, but FTO-2B cells expressed more HNF-3 beta and less HNF-3 alpha, while the reverse was true of H4AzC2 and H5D.7 cells.

Spatial and temporal analyses of integrin and Muc-1 expression in porcine uterine epithelium and trophectoderm in vivo.

The spatial and temporal expressions of Muc-1, selected integrin subunits, and extracellular matrix components in porcine uterine epithelium from estrous (Days 0, 4, 8, 10-15) and early pregnant (Days 10-15 of pregnancy) gilts and from steroid-treated ovariectomized gilts were analyzed using indirect immunofluorescence analyses on cryosectioned tissues to identify potential components of uterine receptivity. Integrin subunit and extracellular matrix expressions were also examined in Day 11-15 conceptuses. Intense Muc-1 staining was detected on apical uterine epithelium on Day 0 but was absent by Day 10 in both cyclic and pregnant gilts. The result of estrogen treatment (E2; 100 micrograms/day for 10 days) was similar to that of the corn oil vehicle control, while treatment with progesterone (P4; 200 mg/day for 10 days) or E2 + P4 decreased Muc-1 staining in ovariectomized gilts. Immunostaining performed with antibodies directed against integrin subunits (alpha 1, alpha 3, alpha 4, alpha 5, alpha v, beta 1, and beta 3) in uterine epithelium revealed low (integrin subunits alpha 1 alpha 3), high (integrin subunits alpha v and beta 3), or modulated (integrin subunits alpha 4, alpha 5, and beta 1) expression, with the lowest expression on Day 0 and maximum expression by Days 10-15. Additionally, no differences due to pregnancy status were detected in staining of uterine epithelium on Days 10-15. Uterine epithelium from steroid-treated ovariectomized gilts had low expression of alpha 4, alpha 5, and beta 1 subunits in the presence of E2 that increased in response to P4 and E2 + P4 treatments. The expression of integrin subunits alpha 3, alpha v and beta 3 was not affected by sex steroids. Trophectoderm also expressed alpha 1, alpha 4, alpha 5, alpha v, beta 1, and beta 3, integrin subunits. Extracellular matrix constituents (fibronectin, vitronectin, laminin, and collagen type IV) were also examined. Fibronectin and vitronectin were present on trophectoderm, but only vitronectin was detected on uterine epithelium. The alpha 4, alpha 5, alpha v, beta 1, and beta 3 integrin subunits, vitronectin, and fibronectin were detected at sites of attachment between uterine epithelial cells and trophectoderm on Days 12-15 of pregnancy. These studies indicate that down-regulation of Muc-1 coincides with the transition of the prereceptive uterus to the receptive uterus. Additionally, the expression of alpha 4, alpha 5, alpha v, beta 1, and beta 3 integrin subunits along with the extracellular matrix components of fibronectin and vitronectin correlates with the time of implantation in swine.

Modulation of alpha 4 beta 1 and alpha 5 beta 1 integrin expression: heterogeneous effects of Q-switched ruby, Nd:YAG, and alexandrite lasers on melanoma cells in vitro.

BACKGROUND AND OBJECTIVE: Integrins of the beta 1 family are cellular adhesion molecules that play an important role in cell attachment and migration by interacting with extracellular matrix molecules. Agents such as hormones, cytokines, and ultraviolet radiation have all been shown to have an integrin modulating potential. The present study indicates that radiation of Q-switched lasers is also able to induce transient changes in integrin expression levels on human melanoma cells in vitro. STUDY DESIGN/MATERIALS AND METHODS: Radiation from Q-switched Ruby (694 nm), Alexandrite (755 nm), and Nd:YAG laser (1,064 nm) with fluences comparable to those that are generally used in treating dermatologic lesions were used to irradiate a subconfluent layer of human melanoma cells. After fixed time intervals, the cells were harvested either to analyse the integrin expression by flow cytometry or to investigate changes in cell attachment, spreading, and migration. RESULTS: It was established that all three types of laser were able to cause a significant downregulation of both the alpha 4 and the common beta 1 integrin subunit. The Alexandrite and Ruby lasers also induced a decrease in alpha 5 expression; however, the cells treated with the Nd:YAG laser showed a marked upregulation of the alpha 5 subunit. The expression of the other beta 1 integrin subunits was shown to be unaltered after laser treatment. Downregulation of the alpha 4 upregulation of the alpha 5 integrin subunit expression resulted in, respectively, decreased and increased attachment and spreading on fibronectin, the extracellular matrix ligand for both the alpha 4 beta 1 and alpha 5 beta 1 integrins. Marked upregulation of the alpha 5 subunit also resulted in a higher migration rate. CONCLUSION: Taken together, these results show that nonlethal doses of Q-switched laser radiation are able to induce changes in cellular behavior in vitro by modulating the integrin expression pattern.