Biomedical subjects

C M DiPersio

Publications and source records attributed to C M DiPersio.

12 recordsLinked to original sources

PubMed2001

Functional comparison of the alpha3A and alpha3B cytoplasmic domain variants of the chicken alpha3 integrin subunit.

Integrin alpha3beta1 can be alternatively spliced to generate alpha3A and alpha3B cytoplasmic domain variants that are conserved among vertebrates. To identify distinct functions of these variants, we transfected cells with intact alpha3 integrins or chimeric receptors. alpha3Abeta1 and alpha3Bbeta1 each localized to focal contacts in keratinocytes on an extracellular matrix rich in laminin-5, to which both are known to bind with high affinity. However, alpha3B accumulated intracellularly in keratinocytes on collagen, suggesting that laminin binding may stabilize alpha3Bbeta1 surface expression. Neither alpha3 cytoplasmic domain affected recruitment of chimeric alpha5 integrins to fibronectin-induced focal contacts, and either substituted for the alpha5 cytoplasmic domain in alpha5beta1-mediated cell migration. However, the alpha5/alpha3B chimera localized to cell-cell borders in MDCK or CHO cells to a lesser extent than did the alpha5/alpha3A chimera. To determine whether the alpha3 cytoplasmic domains conferred distinct localization to a nonintegrin protein, we transfected cells with interleukin-2 receptor (IL-2R) chimeras containing the alpha3 cytoplasmic domains. The IL-2R/alpha3A chimera was expressed efficiently on the cell surface, while the IL-2R/alpha3B chimera accumulated intracellularly. Our findings suggest that the alpha3B cytoplasmic domain harbors a retention signal that is regulated in an intact integrin and can alter cell surface expression and distribution of alpha3beta1.

Alternative Splicing↗

PubMed2000

Mouse keratinocytes immortalized with large T antigen acquire alpha3beta1 integrin-dependent secretion of MMP-9/gelatinase B.

Remodeling of the extracellular matrix during tissue development, wound repair and tumor cell invasion depends on the coordinated regulation of cell adhesion receptors, matrix proteins and enzymes that proteolyse the extracellular matrix. Integrin alpha3beta1 is a major receptor on epidermal keratinocytes for laminin-5 in the cutaneous basement membrane and is required for normal basement membrane organization during skin development. alpha3beta1 is also expressed at high levels in the majority of adherent transformed cells and in most tumors, and it could have similar roles in extracellular matrix remodeling during tumorigenesis and cell invasion. In the present study, we show that alpha3beta1 expression is required in immortalized mouse keratinocytes (MK) for the production of the matrix metalloproteinase MMP-9/gelatinase B, an MMP that is coexpressed with alpha3beta1 in epithelial cell carcinomas and during wound healing, and contributes to the invasive potential of some tumor cells. MMP-9 was expressed in MK cells derived from wild-type mice, but not in MK cells derived from alpha3-null mice. Reconstitution of alpha3beta1 expression in alpha3-null MK cells through transfection with the alpha3 subunit restored MMP-9 secretion, indicating an alpha3beta1-dependent pathway for MMP-9 production. alpha3beta1-dependent expression of MMP-9 was associated with the immortalized phenotype, since nonimmortalized, primary keratinocytes required soluble growth factors, but not alpha3beta1, for efficient expression of MMP-9. Our results suggest that an alpha3beta1-independent pathway(s) for MMP-9 production is suppressed in keratinocytes immortalized with large T antigen, and that an alpha3beta1-dependent pathway is required for sustained production of MMP-9 in the absence of other pathways.

Actins↗

PubMed2000

alpha3beta1 and alpha6beta4 integrin receptors for laminin-5 are not essential for epidermal morphogenesis and homeostasis during skin development.

Continuous regeneration and homeostasis of the stratified epidermis requires coordinated regulation of cell proliferation, cell differentiation, and cell survival. Integrin-mediated cell adhesion to the extracellular matrix has important roles in regulating each of these processes. Integrins alpha3beta1 and alpha6beta4 are both receptors on epidermal keratinocytes for the basement membrane protein laminin-5, the major ligand for epidermal adhesion in mature skin. Ablation in mice of either alpha3beta1 or alpha6beta4, through null mutation of the gene encoding the alpha3, alpha6, or beta4 integrin subunit, results in epidermal blistering of varying severity. Our previous studies showed that, despite blistering, differentiation and stratification of the epidermis appeared essentially normal in mice that lacked either alpha3beta1 or alpha6beta4. However, these studies did not definitively address the specific developmental importance of each integrin, since they may have overlapping and/or compensatory functions. Given the individual importance of alpha3beta1 or alpha6beta4 in maintaining the dermo-epidermal junction in mature skin, we sought to determine the importance of these integrins for embryonic skin development and epidermal morphogenesis. In the current study, we analyzed skin development in mutant embryos that completely lack both integrins alpha3beta1 and alpha6beta4. Although alpha3beta1/alpha6beta4-deficient embryos displayed epidermal blistering by stage E15.5 of development, they also retained regions of extensive epidermal adhesion to the basement membrane through stage E16.5, indicating alternative adhesion mechanisms. Apoptosis was induced in detached epidermis of alpha3beta1/alpha6beta4-deficient embryos, exemplifying vividly the importance of epithelial attachment to the basement membrane for cell survival. However, apoptotic cells were completely absent from attached epidermis of alpha3beta1/alpha6beta4-deficient embryos, showing that epithelial adhesion that occurred independently of alpha3beta1 and alpha6beta4 also protected cells from apoptosis. Remarkably, in the absence of the known laminin-5 binding integrins (alpha3beta1, alpha6beta4, and alpha6beta1), keratinocytes retained the capacity to proliferate in the epidermis, and epidermal stratification and skin morphogenesis appeared normal prior to blister formation. These findings show that while alpha3beta1 and alpha6beta4 are both required for integrity of the dermo-epidermal junction, neither one is essential for epidermal morphogenesis during skin development.

Animals↗

PubMed1998

Novel roles for alpha3beta1 integrin as a regulator of cytoskeletal assembly and as a trans-dominant inhibitor of integrin receptor function in mouse keratinocytes.

Previously we found that alpha3beta1 integrin-deficient neonatal mice develop micro-blisters at the epidermal-dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of alpha3beta1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of alpha3beta1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of alpha3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and alpha-actinin at focal contact sites in the alpha3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to alpha3beta1 deficiency. Apart from the loss of alpha3beta1 there is no change in expression of the other integrins expressed by the alpha3-null keratinocytes. However, in functional assays, alpha3beta1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of alpha3beta1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.

Actins↗

PubMed1997

alpha3beta1 Integrin is required for normal development of the epidermal basement membrane.

Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.

Animals↗

PubMed1995

Self-complementary oligopeptide matrices support mammalian cell attachment.

A new class of ionic self-complementary oligopeptides is described, two members of which have been designated RAD16 and EAK16. These oligopeptides consist of regular repeats of alternating ionic hydrophilic and hydrophobic amino acids and associate to form stable beta-sheet structures in water. The addition of buffers containing millimolar amounts of monovalent salts or the transfer of a peptide solution into physiological solutions results in the spontaneous assembly of the oligopeptides into a stable, macroscopic membranous matrix. The matrix is composed of ordered filaments which form porous enclosures. A variety of mammalian cell types are able to attach to both RAD16 and EAK16 membranous matrices. These matrices provide a novel experimental system for analysing mechanisms of in vitro cell attachment and may have applications in in vivo studies of tissue regeneration, tissue transplantation and would healing.

Amino Acid Sequence↗

PubMed1995

alpha 3A beta 1 integrin localizes to focal contacts in response to diverse extracellular matrix proteins.

In vitro binding assays and inhibition of cell adhesion with monoclonal antibodies have implicated the integrin alpha 3 beta 1 as a receptor for a variety of extracellular ligands. However, reports of alpha 3 beta 1-ligand interactions are inconsistent, and transfection studies have suggested that alpha 3 beta 1 is not sufficient for cell attachment to ligands other than kalinin/laminin 5. We used immunofluorescence to study subcellular localization of the alpha 3A cytoplasmic domain variant in different cultured cell types. Using standard fixation and permeabilization methods, antibodies specific for alpha 3A stained most cell types in a diffuse pattern, consistent with previous reports. Surprisingly, however, chemical cross-linking of integrins to the extracellular matrix and extraction of the cytoskeleton prior to immunofluorescence revealed alpha 3A in focal contacts of most cells tested, suggesting that the cytoplasmic domain was concealed in intact focal contacts by cytoskeletal or other cytoplasmic proteins. The alpha 3A subunit localized to focal contacts in several cell types cultured on fibronectin, kalinin/laminin 5, EHS-laminin/laminin 1, type IV collagen, or vitronectin. In contrast, alpha 5 and alpha V integrins were detected in focal contacts only in cells grown on their known ligands (fibronectin, and fibronectin or vitronectin, respectively). Therefore, our results show that alpha 3A beta 1 responds to a broad spectrum of extracellular ligands. Time course comparisons of the recruitment of alpha subunits from different fibronectin receptors suggested that localization of alpha 3A beta 1 to fibronectin-induced focal contacts was independent of the recruitment of alpha 5 and alpha 4 integrins. However, other studies have shown that alpha 3A beta 1 does not mediate initial cell adhesion to many of the ligands that induced its focal contact localization, including fibronectin. Therefore, we suggest that alpha 3A beta 1 may be a secondary receptor with post-cell-adhesion functions for a broad spectrum of extracellular matrices.

Amino Acid Sequence↗

PubMed1993

Regulation of albumin gene expression in hepatoma cells of fetal phenotype: dominant inhibition of HNF1 function and role of ubiquitous transcription factors.

Two widely used hepatoma cell lines, mouse BW1J and human HepG2, express gene products characteristic of fetal hepatocytes, including serum albumin, whereas reporter genes driven by the albumin promoter are expressed at very low levels compared with highly differentiated hepatoma cells. We have investigated the low albumin promoter activity in BW1J cells to understand differences in liver gene regulation between fetal and adult cells. Addition of the albumin upstream enhancer, or any other fragment of the albumin gene, failed to modify expression of the transfected promoter in BW1J cells. Analysis of cis elements of the albumin promoter showed that, in contrast to highly differentiated H4II cells, in BW1J cells the activity largely depends on ubiquitous transcription factors. Both BW1J and HepG2 cells produce the liver-enriched transcription factor HNF1; dimerization and DNA binding properties are identical to those of liver HNF1, yet the protein fails to show the anticipated transcriptional stimulatory activity. A transfected HNF1 expression vector strongly trans-activates the albumin promoter in HepG2 but only weakly in BW1J cells, and in hybrids (BW1J x Fao), inefficient HNF1 function is dominant. We conclude that hepatoma cells of the fetal phenotype are deficient in the use of HNF1 to drive transcription of the albumin gene and that they harbor a dominant modulator of HNF1 function.

Animals↗

PubMed1991

Extracellular signals that regulate liver transcription factors during hepatic differentiation in vitro.

A complex cell culture environment has been shown to maintain the differentiated state of hepatocytes, yet the mechanisms by which environmental cues selectively maintain liver-specific gene transcription have been unknown. In this paper we show that the hepatic environment regulates the activities of at least three liver-enriched transcription factors, eE-TF, eG-TF/HNF3, and eH-TF, that activate the mouse serum albumin enhancer. eE-TF is a heat-stable factor that has a DNA-binding specificity similar to that of the liver transcription factor C/EBP, but is a distinct protein. eG-TF/HNF3 contributes to the liver-specific transcription of several other serum protein genes. eH-TF binds to a TGTTTGC sequence that occurs at regulatory sites of the albumin promoter, the hepatitis B virus enhancer, and other hepatic genes. eE-TF, eG-TF/HNF3, and eH-TF are regulated by different combinations of the following cell culture conditions: a hormonally defined serum-free medium; an extracellular matrix gel; and a transformation-competent simian virus 40 large T antigen. We propose a regulatory network model to explain how cues from the cell lineage and the extracellular environment coordinately help maintain the activities of transcription factors involved in hepatocyte differentiation.

Animals↗

PubMed1991

The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.

The extracellular matrix (ECM) promotes tissue morphogenesis, cell migration, and the differentiation of a variety of cell types. However, the mechanisms by which ECM causes differentiated gene expression have been unknown. In this report, we show that culturing the hepatocyte-derived cell line H2.35 on an ECM gel changes cell morphology and selectively stimulates the transcription of a subset of liver-specific genes, including serum albumin. Transcriptional activation by ECM also occurs with transfected plasmids bearing the transcriptional enhancer of the albumin gene. ECM substrates of different composition activated the albumin enhancer only when the ECM promoted a cuboidal, differentiated cell morphology. Enhancer activation by the ECM was mediated by two liver transcription factors, HNF3 alpha and eH-TF, which appear to be regulated differently by matrix. Specifically, we found that a collagen gel substratum caused a selective increase in the factor HNF3 alpha at the levels of mRNA accumulation and DNA-binding activity in nuclear extracts, both in H2.35 cells and in the hepatoma cell line HepG2. We conclude that the ECM can stimulate cell differentiation by selectively activating transcriptional regulatory factors and that such regulation occurs coordinately with ECM-promoted changes in cell shape.

Albumins↗

PubMed1990

Site-directed mutagenesis reveals a liver transcription factor essential for the albumin transcriptional enhancer.

A method based on the polymerase chain reaction is described for constructing a clustered set of basepair changes, deletions, or insertions at any site on a DNA fragment. Advantages of the procedure are that virtually every product has the desired sequence alteration and that only a single round of polymerase chain reaction is required. We used this method to demonstrate that the binding of a specific liver nuclear protein, which we call eH-TF, is essential for the function of the enhancer of the mouse albumin gene. The eH-TF binding activity is hepatocyte-specific; it binds to a functional region of the albumin promoter and is distinct from other albumin promoter factors, and part of the eH-TF binding sequence, TGTTTGC, occurs in functional regulatory sites of other liver-specific genes.

Animals↗

PubMed1988

Conditional enhancement of liver-specific gene transcription.

We sought to develop a cell line in which liver-specific transcription could be induced at will, to facilitate the study of factors that cause hepatocyte-specific transcription of the serum albumin gene in mice. We therefore created the H2.35 cell line from mouse hepatocytes infected with a temperature-sensitive strain of simian virus 40. During routine propagation at the permissive temperature, H2.35 cells exhibit extremely low levels of albumin transcription and mRNA. Albumin mRNA increases at least 100-fold when H2.35 cells are cultured at the restrictive temperature and in serum-free medium on a collagen substratum; the two latter conditions maintain the differentiated state of primary hepatocyte cultures. Although a major cause of the mRNA increase is posttranscriptional, the transcription rates of albumin and other liver-specific genes increase significantly. Transient-transfection experiments demonstrated that an induction of transcription is caused by activation of an albumin upstream sequence that was previously shown to enhance liver-specific transcription in transgenic mice. Thus, hepatocyte differentiation appears to be maintained in part by extracellular signals that stimulate the activity of a tissue-specific enhancer element.

Animals↗