CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

BACKGROUND: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. METHODS: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). RESULTS: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. CONCLUSIONS: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

Postprandial glucose and HbA1c are associated with severity of obstructive sleep apnoea in non-diabetic obese subjects.

INTRODUCTION: Obstructive sleep apnoea (OSA) is an underdiagnosed condition frequently associated with glycaemic control impairment in patients with type 2 diabetes. AIM: To assess the relationship between glycometabolic parameters and OSA in obese non-diabetic subjects. METHODS: Ninety consecutive subjects (mean age 44.9 ± 12 years, mean BMI 42.1 ± 9 kg/m2) underwent polysomnography and a 2-h oral glucose tolerance test (OGTT). RESULTS: OSA was identified in 75% of subjects, with a higher prevalence of males compared to the group of subjects without OSA (62% vs 32%, p = 0.02). Patients with OSA had comparable BMI (42.8 kg/m2 vs 39.4 kg/m2), a higher average HbA1c (5.8% vs 5.4%, p < 0.001), plasma glucose at 120 min during OGTT (2 h-PG; 123 mg/dl vs 97 mg/dl, p = 0.009) and diastolic blood pressure (81.1 mmHg vs 76.2 mmHg, p = 0.046) than obese subjects without OSA. HbA1c and 2 h-PG were found to be correlated with the apnoea-hypopnoea index (AHI; r = 0.35 and r = 0.42, respectively) and with percent of sleep time with oxyhaemoglobin saturation < 90% (ST90; r = 0.44 and r = 0.39, respectively). Further, in a linear regression model, ST90 and AHI were found to be the main determinants of 2 h-PG (ß = 0.81, p < 0.01 and ß = 0.75, p = 0.02, respectively) after controlling for age, sex, waist circumference, physical activity, and C-reactive protein. Similarly, ST90 and AHI persisted as independent determinants of HbA1c (ß = 0.01, p = 0.01 and ß = 0.01, p = 0.01, respectively). CONCLUSION: Beyond the traditional clinical parameters, the presence of a normal-high value of 2 h-PG and HbA1c should raise suspicion of the presence of OSA in obese subjects.

Vitamin D deficiency as a predictor of poor prognosis in patients with acute respiratory failure due to COVID-19.

PURPOSE: Hypovitaminosis D is a highly spread condition correlated with increased risk of respiratory tract infections. Nowadays, the world is in the grip of the Coronavirus disease 19 (COVID 19) pandemic. In these patients, cytokine storm is associated with disease severity. In consideration of the role of vitamin D in the immune system, aim of this study was to analyse vitamin D levels in patients with acute respiratory failure due to COVID-19 and to assess any correlations with disease severity and prognosis. METHODS: In this retrospective, observational study, we analysed demographic, clinical and laboratory data of 42 patients with acute respiratory failure due to COVID-19, treated in Respiratory Intermediate Care Unit (RICU) of the Policlinic of Bari from March, 11 to April 30, 2020. RESULTS: Eighty one percent of patients had hypovitaminosis D. Based on vitamin D levels, the population was stratified into four groups: no hypovitaminosis D, insufficiency, moderate deficiency, and severe deficiency. No differences regarding demographic and clinical characteristics were found. A survival analysis highlighted that, after 10 days of hospitalization, severe vitamin D deficiency patients had a 50% mortality probability, while those with vitamin D ≥ 10 ng/mL had a 5% mortality risk (p = 0.019). CONCLUSIONS: High prevalence of hypovitaminosis D was found in COVID-19 patients with acute respiratory failure, treated in a RICU. Patients with severe vitamin D deficiency had a significantly higher mortality risk. Severe vitamin D deficiency may be a marker of poor prognosis in these patients, suggesting that adjunctive treatment might improve disease outcomes.

Influence of circadian rhythm on exhaled breath profiling by electronic nose.

Electronic noses (e-noses) are a cheap and easy method for exhaled Volatile Organic Compound (VOC)-analysis which has shown its potential in several diseases. Before obtaining a full validation of these instruments in clinical settings, a number of methodological issues still have to be established. We aimed to investigate a potential influence of circadian variation on VOC-profile analyzed by an e-nose in healthy subjects. We enrolled 22 adults free of any known diseases. A sequence of exhaled breath samplings were performed on all participants at predetermined hours (7am, 12pm, 17pm, 23pm) and analyzed by an e-nose (Cyranose 320). According to Principal Component Analysis, significant circadian variations of the exhaled VOC-profile were shown for Principal Component (PC) 1 and 3. In detail, PC1 and PC3 values were significantly higher in the morning compared to the afternoon and evening (for all parameters p less than 0.05). Successive Linear Discriminant analysis confirmed the findings above. The daily variations in VOCs-profile, with the peak in the morning, could be relevant for future clinical applications, especially in the choice of optimal time for sampling patients.

Standardization procedure for the nasal nitric oxide measurement method using Niox MINO® and the tidal-breathing technique with velum-closure.

Nitric oxide (NO) is a molecule that performs many functions in the human body. The entire respiratory tract can produce NO, but the highest production occurs in the upper respiratory tract, in the paranasal sinuses in particular. The aim of the present study was to assess a new nasal NO (nNO) measurement method using the Niox MINO Nasal® device (Aerocrine AB, Solna, Sweden) and a special procedure, in order to compare the nNO values obtained in 32 healthy subjects with the values found in the international literature. The measured normal nNO values were equal to 426.76±143.27 ppb, with a 95% confidence interval [160.22-733.30]. Males had an average nNO value equal to 446.76±133.63 [178.64 – 714.02], whereas in females the average value was 403.80±154.90 [94.00-713.60]. This study allows us to confirm that we have been able to establish the normal range of nitric oxide quantity produced in the nasal/sinus cavities of healthy individuals using the Niox MINO Nasal® device and tidal-breathing with velum-closure manoeuvre.

Derivation and experimental comparison of cell-division probability densities.

Experiments have shown that, even in a homogeneous population of cells, the distribution of division times is highly variable. In addition, a homogeneous population of cells will exhibit a heterogeneous response to drug therapy. We present a simple stochastic model of the cell cycle as a multistep stochastic process. The model, which is based on our conception of the cell cycle checkpoint, is used to derive an analytical expression for the distribution of cell cycle times. We demonstrate that this distribution provides an accurate representation of cell cycle time variability and show how the model relates drug-induced changes in basic biological parameters to variability in response to drug treatment.

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

Background: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. Methods: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven Braf V600E /Pten -/- /Cxcr2 -/- and NRas Q61R /INK4a -/- /Cxcr2 -/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in Braf V600E /Pten -/- and NRas Q61R /INK4a -/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). Results: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1 , a key tumor suppressive transcription factor, was the only gene significantly induced with a log 2 fold-change greater than 2 in these three different melanoma models. Conclusions: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

Chloroquine affects biosynthesis of Ia molecules by inhibiting dissociation of invariant (gamma) chains from alpha-beta dimers in B cells.

Biosynthetic conversion of Ia oligomers from three chains (alpha, beta, gamma) to two (alpha, beta) before surface expression was inhibited in B lymphoid cells by treatment with chloroquine, resulting in the accumulation of Ia complexes composed of mature alpha and beta chains, and gamma chains at various states of sialylation. Other stages of Ia biosynthesis and processing appeared unaffected, indicating that chloroquine selectively interfered with the gamma chain dissociating mechanism itself. Similar effects were also observed with ammonium chloride. Because of the nature of such lysosomotropic agents, these results suggest that an intracellular acidic compartment may be involved in processing Ia oligomers to accomplish dissociation from gamma chains. Since chloroquine is known to inhibit Ia-restricted antigen presentation in accessory cells, our results raise the possibility that the pathways of antigen processing and Ia biosynthesis may use some common intracellular compartments.

Four Ia invariant chain forms derive from a single gene by alternate splicing and alternate initiation of transcription/translation.

We determined the structural basis for the presence of electrophoretically-distinct, antigenically-related forms of invariant chains in Ia oligomers, and established the mechanisms by which they can be expressed from a single gene. S1 nuclease protection assays indicated that, in B cells, transcription of this gene initiates at a minimum of three sites. Thus, unlike previously thought, invariant chain mRNAs have heterogeneous 5' untranslated segments that may differentially affect initiation of translation. Further, restriction mapping and nucleotide sequencing of cDNAs revealed two kinds of invariant chain mRNAs differing by an internal coding segment of 192 bp. This segment represents an alternatively spliced exon, as demonstrated by nucleotide sequencing of corresponding genomic regions. The exon (exon X) encodes a cysteine-rich stretch of 64 amino acids near the COOH terminus that displays a striking and surprising homology to an internal amino acid repeat of thyroglobulin, suggesting an evolutionary mechanism of exon shuffling. Transient expression of cDNAs indicated that both types of alternatively spliced mRNAs contain two in-frame AUGs functioning as alternate start sites for translation. Thus, transfections with exon X-lacking cDNAs resulted in the expression of Mr 33,000 and 31,000 proteins, detected by immunoprecipitation with anti-invariant chain antisera, and identical by two-dimensional gel (2-D) analyses to the B cell invariant-chain forms gamma 1 (Mr 31,000), gamma 2, and gamma 3 (Mr 33,000). Similarly, exon X-containing cDNAs expressed Mr 43,000 and 41,000 proteins, also identical by 2-D migration to Ia-associated proteins. Thus, human Ia molecules contain four forms of invariant chain of closely related but nonidentical primary structure that are generated from a single gene by a complex pattern of alternate transcriptional start, exon splicing, and translational start.

A recurrent idiotype on monoclonal anti-human Ia antibodies.

We report a recurrent idiotype on a remarkably high fraction (4/19) of murine monoclonal antibodies specific for human Ia monomorphic determinants and elicited by separate immunizations. For three of them, the shared idiotype is associated with the antigen-combining site. These results indicate that the spectrum of mouse antibody responses to human Ia antigens may be based on recurrent idiotypes, suggesting a limited potential repertoire of murine monoclonal antibodies to human Ia antigens. Anti-idiotypic reagents might be helpful in dissecting this repertoire and to generate a mirror image of a human Ia antigenic map. Furthermore, antisera to the idiotype of antibodies specific for human Ia monomorphic determinants might help in elucidating the interactions between Ia molecules and receptors on immune cells.

The human invariant chain is the core protein of the human class II-associated proteoglycan.

The human class II-associated chondroitin sulfate proteoglycan (CSPG) was analyzed biochemically and immunologically to determine a possible relationship with the human invariant chain (gamma 1) and its related components. The CSPG was purified by a three-step procedure involving associative ion-exchange chromatography, immunoprecipitation, and dissociative ion-exchange chromatography. Treatment of the CSPG with chondroitinase revealed core proteins of Mr approximately 46,000, 38,000, and 28,000, with the 38,000 species most highly represented. Tryptic peptide analysis revealed identity of the peptides of the 38,000 Mr core protein and gamma 1, and of the 28,000 Mr species and p25. The CSPG and its core proteins were shown to react directly with the mouse anti-human invariant chain monoclonal antibody VIC-Y1 and a rabbit antiserum produced against a synthetic peptide corresponding to the C-terminal end of invariant chain. These results demonstrate that the invariant chain is the core protein of the class II-associated CSPG. In addition, virtually all the CSPG was shown to be present on the cell surface.

The internal affairs of an integrin.

Integrins are adhesion receptors that exchange signals between the extracellular and intracellular compartments. From their cell surface transmembrane location, they interact with extracellular matrix ligands or cellular counter-receptors, translating external cues into signals that affect cytoskeletal organization, cell shape and motility. Conversely, intracellular events may modify the affinities of integrins for external ligands. Inside the cell, integrins connect with cytoskeletal structures that, until recently, were thought to be exclusively actin microfilaments. We comment on the case of the epithelial integrin, alpha(6)beta(4), which may instead interact with intermediate filaments. This integrin was recently shown by several laboratories to be part of the hemidesmosome complex, an epithelial adhesive structure that is the plasma membrane anchoring site for keratin-containing intermediate filaments.

The major laminin receptor of mouse embryonic stem cells is a novel isoform of the alpha 6 beta 1 integrin.

Laminin is the first extracellular matrix protein expressed in the developing mouse embryo. It is known to influence morphogenesis and affect cell migration and polarization. Several laminin receptors are included in the integrin family of extracellular matrix receptors. Ligand binding by integrin heterodimers results in signal transduction events controlling cell motility. We report that the major laminin receptor on murine embryonic stem (ES) cells is the integrin heterodimer alpha 6 beta 1, an important receptor for laminin in neurons, lymphocytes, macrophages, fibroblasts, platelets and other cell types. However, the cytoplasmic domain of the ES cell alpha 6 (alpha 6 B) differs totally from the reported cytoplasmic domain amino acid sequence of alpha 6 (alpha 6 A). Comparisons of alpha 6 cDNAs from ES cells and other cells suggest that the alpha 6 A and alpha 6 B cytoplasmic domains derive from alternative mRNA splicing. Anti-peptide antibodies to alpha 6 A are unreactive with ES cells, but react with mouse melanoma cells and embryonic fibroblasts. When ES cells are cultured under conditions that permit their differentiation, they become positive for alpha 6 A, concurrent with the morphologic appearance of differentiated cell types. Thus, expression of the alpha 6 B beta 1 laminin receptor may be favored in undifferentiated, totipotent cells, while the expression of alpha 6 A beta 1 receptor occurs in committed lineages. While the functions of integrin alpha chain cytoplasmic domains are not understood, it is possible that they contribute to transferring signals to the cell interior, e.g., by delivering cytoskeleton organizing signals in response to integrin engagement with extracellular matrix ligands. It is therefore reasonable to propose that the cellular responses to laminin may vary, according to what alpha subunit isoform (alpha 6 A or alpha 6 B) is expressed as part of the alpha 6 beta 1 laminin receptor. The switch from alpha 6 B to alpha 6 A, if confirmed in early embryos, could then be of striking potential relevance to the developmental role of laminin.

Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing.

A transmembrane extracellular matrix receptor of the integrin family, alpha 6 beta 4, is a component of the hemidesmosome, an adhesion complex of importance in epithelial cell-connective tissue attachment (Stepp, M. A., S. Spurr-Michaud, A. Tisdale, J. Elwell, and I. K. Gipson. 1990. Proc. Natl. Acad. Sci. USA. 87:8970-8974; Jones, J. C. R., M. A. Kurpakus, H. M. Cooper, and V. Quaranta. 1991. Cell Regulation. 2:427-438). Cytosolic components of hemidesmosomes include bullous pemphigoid (BP) antigens while extracellular components include a 125-kD component of anchoring filaments (CAF) and collagen type VII-containing anchoring fibrils. We have monitored the incorporation of the alpha 6 beta 4 integrins into forming hemidesmosomes in an in vitro wound-healing explant model. In epithelial cells recently migrated from the edges of unwounded sites over bare connective tissue, alpha 6 beta 4 first appears along the entire cell surface. At this stage, these cells contain little or no cytosolic hemidesmosomal components, at least as detectable by immunofluorescence using BP autoantibodies, whereas they are already positive for laminin and CAF. At a later stage, as cells become positive for cytosolic hemidesmosome components such as BP antigens as well as collagen type VII, alpha 6 beta 4 becomes concentrated along the basal pole of the epithelial cell where it abuts the connective tissue of the explant. Polyclonal antibodies to beta 4 do not interfere with the migration of epithelial cells in the explant. However, they prevent assembly of hemidesmosomal complexes and inhibit expression of collagen type VII in cells that have migrated over wound areas. In addition, they induce disruption of established hemidesmosomes in nonmigrating cells of the unwounded area of the explant. Monoclonal antibodies to alpha 6 have a more dramatic effect, since they completely detach epithelial cells in the unwounded area of the explant. Antibodies to CAF also detach epithelial cells in unwounded areas, apparently by inducing separation between epithelium and connective tissue at the lamina lucida of the basement membrane zone. These results suggest a model whereby polarization of alpha 6 beta 4 to the basal surface of the cells, perhaps induced by a putative anchoring filament-associated ligand, triggers assembly of hemidesmosome plaques.

Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5.

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.

Laminin receptors in the retina: sequence analysis of the chick integrin alpha 6 subunit. Evidence for transcriptional and posttranslational regulation.

The integrin alpha 6 beta 1 is a prominent laminin receptor used by many cell types. In the present work, we isolate clones and determine the primary sequence of the chick integrin alpha 6 subunit. We show that alpha 6 beta 1 is a prominent integrin expressed by cells in the developing chick retina. Between embryonic days 6 and 12, both retinal ganglion cells and other retinal neurons lose selected integrin functions, including the ability to attach and extend neurites on laminin. In retinal ganglion cells, we show that this is correlated with a dramatic decrease in alpha 6 mRNA and protein, suggesting that changes in gene expression account for the developmental regulation of the interactions of these neurons with laminin. In other retinal neurons the expression of alpha 6 mRNA and protein remains high while function is lost, suggesting that the function of the alpha 6 beta 1 heterodimer in these cells is regulated by posttranslational mechanisms.

Laminin 5 in the human thymus: control of T cell proliferation via alpha6beta4 integrins.

Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.

Epithelial integrin alpha 6 beta 4: complete primary structure of alpha 6 and variant forms of beta 4.

The integrin alpha 6 beta 4 is a heterodimer predominantly expressed by epithelia. While no definite receptor function has yet been assigned to it, this integrin may mediate adhesive and/or migratory functions of epithelial cells. We have determined the complete primary structure of both the alpha 6 and beta 4 subunits from cDNA clones isolated from pancreatic carcinoma cell line libraries. The deduced amino acid sequence of alpha 6 is homologous to other integrin alpha chains (18-26% identity). Antibodies to an alpha 6 carboxy terminus peptide immunoprecipitated alpha 6 beta 4 complexes from carcinoma cells and alpha 6 beta 1 complexes from platelets, providing further evidence for the association of alpha 6 with more than one beta subunit. The deduced amino acid sequence of beta 4 predicts an extracellular portion homologous to other integrin beta chains, and a unique cytoplasmic domain comprised of greater than 1,000 residues. This agrees with the structures of the beta 4 cDNAs from normal epithelial cells (Suzuki, S., and Y. Naitoh. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:757-763; Hogervost, F., I. Kuikman, A. E. G. Kr. von dem Borne, and A. Sonnenberg. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:765-770). Compared to these structures, however, the beta 4 cDNAs that we have cloned from carcinoma cells contain extra sequences. One of these is located in the 5'-untranslated region, and may encode regulatory sequences. Another specifies a segment of 70 amino acids in the cytoplasmic tail. Amplification by reverse transcription-polymerase chain reaction of mRNA indicated that multiple forms of beta 4 may exist, possibly due to cell-type specific alternative splicing. The unique structure of beta 4 suggests its involvement in novel cytoskeletal interactions. Consistent with this possibility, alpha 6 beta 4 is mostly concentrated on the basal surface of epithelial cells, but does not colocalize with components of adhesion plaques.

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.