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R Cancedda

Publications and source records attributed to R Cancedda.

At least 37 records · Page 2Linked to original sources
PubMed1995

Chondrocyte and osteoblast differentiation stage-specific monoclonal antibodies as a tool to investigate the initial bone formation in developing chick embryo.

Taking advantage of monoclonal antibodies raised against chick hypertrophic chondrocytes (BD5, LA5, AD2) and osteoblasts (SB1, SB2, SB3, SB5), we have investigated expression of differentiation stage-specific antigens by further differentiating hypertrophic chondrocytes. Expression of all antigens was developmentally regulated during in vitro further differentiation of hypertrophic chondrocytes and all monoclonal antibodies, including those raised against osteoblasts, recognized antigens synthesized by chondrocytes at specific differentiation stages. In particular, the expression of the SB5 antigen, considered specific for secretory osteoblasts incorporated into the bone matrix, progressively increased during the chondrocyte culture and reached its maximum at the time of matrix mineralization. Interestingly, the SB3 antigen was transiently expressed at an intermediate phase of the culture with a staining pattern characteristic of cell-cell adhesion proteins. When cryosections of the chondro-bone junction of developing embryo tibiae were stained, it was observed that monoclonal antibodies raised against hypertrophic chondrocytes stained frank hypertrophic and elongated chondrocytes (BD5, LA5), a very discrete peripheric layer of cartilage (AD2), and cells embedded in the initial osteoid matrix (LA5), but failed to stain cells in the preosteoblast and osteoblast region. On the contrary, monoclonal antibodies recognizing antigens on the surface of cells in the osteogenic layer surrounding the initial bone, also stained frank hypertrophic and/or elongated chondrocytes on the cartilage side of the initial bone (SB1, SB2, SB3). The SB5 monoclonal antibody, in addition to staining cells in the osteoid, also stained a few chondrocytes immediately adjacent. These observations support the concept that hypertrophic chondrocytes may further differentiate to osteoblast-like cells and participate to the initial bone formation.

Animals↗
PubMed1995

Expression of the cartilage matrix protein gene at different chondrocyte developmental stages.

Cartilage matrix protein (CMP), a major noncollagenous component of certain types of hyaline cartilage, is synthesized by chondrocytes in a developmentally regulated manner. In this study, we monitored the accumulation of CMP in the developing chicken limb and sternum by immunostaining. In older embryos, the specific extracellular staining was restricted to the resting/proliferative zone of metaphyseal cartilage and to the immediately adjacent hypertrophic cartilage. A lack of staining was observed in the peripheral layers of articular cartilage. Data were compared with the accumulation of CMP mRNA measured by Northern analysis relative to other cartilage-specific messages in cell cultures representing different stages of chondrocyte differentiation, as well as with the steady state mRNA levels in tissue samples. We found a correlation between the gene expression pattern of the in vitro cultures and the one observed in certain in vivo differentiation stages. The high-density mesenchyme culture was utilized as a model for studying the events at early stage I (stage Ia) of chondrogenesis. This culture was characterized by relatively low steady state mRNA levels for cartilage proteins, including the later activation of the CMP gene as compared to type II collagen or link protein genes, and relatively high steady state mRNA levels for type VI collagen and beta-actin. Chicken embryo chondrocyte cultures obtained from sterna of 14-day-old embryos, however, consisted predominantly of stage Ib chondrocytes, and showed high steady state levels for cartilage proteins, but relatively lower levels for type VI collagen and beta-actin mRNAs. In accordance with the in vivo data, a relatively high steady state level was detected for CMP mRNA in cultures of hypertrophic (stage II) chondrocytes. We also performed transient expression assays in the various culture systems to study the role of the promoter upstream and intronic control regions in the tissue- and developmental stage-specific regulation of the CMP gene. We showed that the enhancer worked in a lineage-specific manner, by further stimulating the minimal promoter activity independent of the developmental stage of chondrocytes, while it did not in other tissues. The promoter upstream control regions, however, seemed to play a role in restricting the promoter activity to a certain chondrocyte developmental stage.

Animals↗
PubMed1994

Hypertrophic chondrocytes undergo further differentiation to osteoblast-like cells and participate in the initial bone formation in developing chick embryo.

Differentiation of hypertrophic chondrocytes to an osteoblast-like phenotype occurs in vivo in the hypertrophic cartilage of chick embryo tibiae underneath early or prospective periosteum and in cartilage around vascular canals. Synthesis of type I collagen by hypertrophic chondrocytes was shown by immunolocalization of the C propeptide. By enzyme cytochemistry it was instead shown that, in vivo, further differentiating hypertrophic chondrocytes express alkaline phosphatase at the time of initial mineral deposition. Evidence that hypertrophic chondrocytes may resume proliferation was obtained by BrdU labeling. A monoclonal antibody (LA5) was isolated and characterized that recognizes a hypertrophic chondrocyte membrane protein. In addition to staining hypertrophic chondrocytes surrounded by a type II and type X collagen-stainable matrix, the LA5 antibodies also stained elongated chondrocytes at the cartilage/bone collar interface and cells incorporated in the first layer of bone and osteoid matrix.

Animals↗
PubMed1994

N-CAM and N-cadherin expression during in vitro chondrogenesis.

Mesenchymal cell condensation in chick limb bud occurs at embryonic stage 22 and is the starting event of chondrogenesis. Several mechanisms have been proposed to have an active role in the induction of this process. Among them the establishment of cell-cell contacts represents a key event. Here we have investigated the modulation of N-CAM and N-cadherin gene expression in an in vitro culture system which allows chondrocyte differentiation to proceed from condensation of prechondrogenic cells to hypertrophic chondrocytes and eventually to osteoblast-like cells. Both Northern and Western blots demonstrated that they were developmentally regulated in differentiating chondrocytes. Both cell adhesion proteins were detectable in prechondrogenic cells, increased during cell aggregation, became undetectable in hypertrophic chondrocytes, and resulted in reexpression during their maturation to osteoblast-like cells. The timing of appearance of N-cadherin and N-CAM suggests that N-cadherin initiates the in vitro cell condensation thereafter stabilized by N-CAM. In agreement with the above findings, the immunolocalization of these molecules in the cell aggregates revealed that N-CAM and N-cadherin appear, after 12 h of suspension culture, on the surface of all cells at the membrane regions participating in cell-cell contacts. At 72 h N-CAM became restricted to cells at the aggregate periphery, while N-cadherin was detected both in type II collagen-negative and -positive regions. At this time of culture, electron microscopy shows a number of cell-cell contacts at the perifery of the cell aggregates, while only a few of them were observed in the aggregate interior. The expression of N-CAM and type II collagen by chondrocytes was mutually exclusive and a sorting out between differentiating and nondifferentiating cells occurred.

Animals↗
PubMed1994

Ovotransferrin and ovotransferrin receptor expression during chondrogenesis and endochondral bone formation in developing chick embryo.

Ovotransferrin expression during chick embryo tibia development has been investigated in vivo by immunocytochemistry and in situ hybridization. Ovotransferrin was first observed in the 7 day cartilaginous rudiment. At later stages, the factor was localized in the articular zone of the bone epiphysis and in the bone diaphysis where it was concentrated in hypertrophic cartilage, in zones of cartilage erosion and in the osteoid at the chondro-bone junction. When the localization of the ovotransferrin receptors was investigated, it was observed that chondrocytes at all stages of differentiation express a low level of the oviduct (tissue) specific receptor. Interestingly, high levels of the receptor were detectable in the 13-d old tibia in the diaphysis collar of stacked-osteoprogenitor cells and in the layer of derived osteoblasts. High levels of oviduct receptor were also observed in the primordia of the menisci. Metabolic labeling of proteins secreted by cultured chondrocytes and osteoblasts and Northern blot analysis of RNA extracted from the same cells confirmed and completed the above information. Ovotransferrin was expressed by in vitro differentiating chondrocytes in the early phase of the culture and, at least when culture conditions allowed extracellular matrix assembly, also by hypertrophic chondrocytes and derived osteoblast-like cells. Osteoblasts directly obtained from bone chips produced ovotransferrin only at the time of culture mineralization. By Western blot analysis, oviduct receptor proteins were detected at a very low level in extract from differentiating and hypertrophic chondrocytes and at a higher level in extract from hypertrophic chondrocytes undergoing differentiation to osteoblast-like cells and from mineralizing osteoblasts. Based on these results, the existence of autocrine and paracrine loops involving ovotransferrin and its receptor during chondrogenesis and endochondral bone formation is discussed.

Animals↗
PubMed1993

Molecular cloning of trkE, a novel trk-related putative tyrosine kinase receptor isolated from normal human keratinocytes and widely expressed by normal human tissues.

We have identified and cloned a new member of the trk gene family, termed trkE, which generates a 3.9-kilobase (kb) transcript in normal human keratinocytes and in a variety of normal human tissues, but not in liver. Albeit at low level, trkE transcript is expressed also by PC12 cells. The open reading frame codes for a polypeptide of 876 amino acids exhibiting the classic features of cell surface tyrosine protein kinases. trkE catalytic domain is 41% identical to trkA and shows several features unique to the trk gene family. Its extracellular domain does not show significant homology to any known proteins. trkE is the first member of this gene family found abundantly and widely expressed in normal human tissues. Several lines of evidence suggest that NGF is also the ligand for trkE; (i) normal human keratinocytes bind NGF with high affinity, (ii) NGF stimulates keratinocyte growth in an autocrine fashion, (iii) NGF exerts its biological effect on keratinocytes through the stimulation of a trk-specific tyrosine kinase, and (iv) keratinocytes lack trkA but do express large amount of trkE. trkE might also be the NGF receptor by other human peripheral tissues, such as pancreatic islets, and might represent a non-neuronal receptor for this ligand.

Amino Acid Sequence↗
PubMed1993

Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.

Normal human keratinocytes synthesize and secrete biologically active nerve growth factor (NGF) in a growth regulated fashion (Di Marco, E., Marchisio, P. C., Bondanza, S., Franzi, A. T., Cancedda, R., and De Luca, M. (1991) J. Biol. Chem. 266, 21718-21722). Here we show that the same human keratinocytes bind NGF via low and high affinity receptors. In parallel with the course of NGF synthesis, the expression of low affinity NGF receptor (p75NGFr) decreases when a confluent, differentiated, and fully stratified epithelium is obtained. In skin sections, p75NGFr is present in basal keratinocytes and absent from suprabasal, terminally differentiated cells. The trkA protooncogene product (p140trkA), a component of the NGF receptor, is not expressed by keratinocytes. Instead, keratinocytes express a new member of the trk family (that we termed trkE), which generates 3.9-kilobase transcripts. Keratinocyte-derived NGF plays a key role in the autocrine epidermal cell proliferation. This has been proven by (i) direct effect of NGF on [3H]thymidine incorporation, (ii) inhibition of autocrine keratinocyte growth by monoclonal antibodies (alpha D11) inhibiting human NGF biological activity, and (iii) inhibition of autocrine keratinocyte proliferation by a trk-specific inhibitor, the natural alkaloid K252a. These data provide evidence that NGF, in addition to its effect as a survival and differentiation factor, is a potent regulator of cell proliferation, at least in human epithelial cells.

3T3 Cells↗
PubMed1993

Modulation of tensin and vimentin expression in chick embryo developing cartilage and cultured differentiating chondrocytes.

It has been proposed that tensin, in association with several other proteins, mediates the micro-filament-integrin link. Here we describe the isolation of clones spanning about 5 kb from the 3' end of tensin mRNA from cultured chick embryo chondrocyte and embryonic heart cDNA libraries. Tensin expression was investigated in cultured chick embryo cells. It was observed that tensin expression is dependent upon substrate adhesion and it is turned off after 7 days of suspension culture. This process is reversible. Tensin expression is also regulated during cartilage cell differentiation in vivo; at Hamburger and Hamilton stage 39-40, non-hypertrophic tibial chondrocytes express both RNA and protein while hypertrophic chondrocytes do not. In the culture system the expression of vimentin, a major component of intermediate filaments, showed an opposite behaviour since the suspension culture enhances the accumulation of both vimentin and its mRNAs. Therefore in chick embryo cultured chondrocytes and in vivo, during cartilage development, cell shape changes and/or integrin-extracellular matrix protein interactions may be involved in the regulation of these two genes coding for cytoskeletal proteins.

Amino Acid Sequence↗
PubMed1993

Type XIV collagen is encoded by alternative transcripts with distinct 5' regions and is a multidomain protein with homologies to von Willebrand's factor, fibronectin, and other matrix proteins.

The combined nucleotide sequences of several overlapping cDNAs provide the first complete amino acid sequence of type XIV collagen. Independent confirmation of the deduced sequence is provided by amino acid sequencing of several tryptic peptides isolated from purified chicken skin type XIV collagen. Comparative analyses show that the amino-terminal non-triple-helical region of alpha 1(XIV) chains contains sequence motifs that are similar to alpha 1(IX) collagen, fibronectin type III repeats, and von Willebrand's factor A-domains. The results also strongly suggest that the alpha 1(XIV) collagen gene is identical to the gene encoding the matrix component previously named undulin. cDNAs covering the 5' region of alpha 1(XIV) mRNA fall into two classes with distinct sequences in their 5'-untranslated regions. We believe the two alternative sequences result from differential splicing of the primary transcript. Interestingly, one of the untranslated sequences shows a high degree of identity with the cis-regulatory translational control sequence in the 5'-untranslated region of a Drosophila ribosomal protein mRNA. We hypothesize therefore that the sequence in alpha 1(XIV) collagen may play a role in the control of alpha 1(XIV) protein synthesis.

Alternative Splicing↗
PubMed1993

The basement membrane protein BM-600/nicein codistributes with kalinin and the integrin alpha 6 beta 4 in human cultured keratinocytes.

The observation is reported that in low-passage human keratinocyte colonies cultured under conditions that allow full epidermal differentiation (i) the basement membrane protein BM-600/nicein, identified by the mAb GB3, is codistributed with laminin and collagen type IV as well as with the bullous pemphigoid antigen in footprints deposited by growing and migrating colonies; (ii) the integrin heterodimer alpha 6 beta 4 is codistributed with the same molecules suggesting its spatial association with basement membrane components; (iii) the distribution pattern of alpha 6 beta 4 and BM-600/nicein underneath individual cells is identical and is characterized by a typical "leopard skin" pattern complementary to the distribution of submembraneous F-actin microfilament network; (iv) a rabbit polyclonal antiserum to kalinin (R4012) used in double-label immunofluorescence staining with mAb GB3 shows that this protein has the same distribution as BM-600/nicein and this suggests that they are identically located; and (v) immunoprecipitation with mAb GB3 to BM-600/nicein and BM165 to kalinin shows identical bands suggesting that nicein and kalinin represent the same molecular entity. We suggest that alpha 6 beta 4 displays not only an adhesive role for keratinocytes in view of its reported association to hemidesmosomes but may also be involved in organizing the molecules of the epithelial extracellular matrix, including those forming the basement membrane zone and hemidesmosomes, a function proposed for other integrins in other cellular systems.

Actins↗
PubMed1993

One-step treatment of proximal hypospadias by the autologous graft of cultured urethral epithelium.

Surgical management of severe proximal hypospadias or long strictures of the posterior urethra is a difficult clinical task. Often, the therapeutic approach involves the autologous graft of free flaps of bladder or oral mucosa. We recently reported the use of autologous graft of cultured squamous urethral epithelium during urethroplasty in patients with severe proximal hypospadias. The main limitation to the widespread use of cultured epithelium was the long hospitalization due to the requirement of 2 surgical steps. We now report a substantial modification of the surgical procedure which allows for rapid 1-step urethroplasty. Cultured squamous urethral epithelium is tubularized in vitro with the aid of a tubular polytetrafluoroethylene (Gore-Tex) support and 1-step urethroplasty is performed within 30 minutes. Results obtained in 8 patients are presented.

Adolescent↗
PubMed1993

Cell proliferation, extracellular matrix mineralization, and ovotransferrin transient expression during in vitro differentiation of chick hypertrophic chondrocytes into osteoblast-like cells.

Differentiation of hypertrophic chondrocytes toward an osteoblast-like phenotype occurs in vitro when cells are transferred to anchorage-dependent culture conditions in the presence of ascorbic acid (Descalzi Cancedda, F., C. Gentili, P. Manduca, and R. Cancedda. 1992. J. Cell Biol. 117:427-435). This process is enhanced by retinoic acid addition to the culture medium. Here we compare the growth of hypertrophic chondrocytes undergoing this differentiation process to the growth of hypertrophic chondrocytes maintained in suspension culture as such. The proliferation rate is significantly higher in the adherent hypertrophic chondrocytes differentiating to osteoblast-like cells. In cultures supplemented with retinoic acid the proliferation rate is further increased. In both cases cells stop proliferating when mineralization of the extracellular matrix begins. We also report on the ultrastructural organization of the osteoblast-like cell cultures and we show virtual identity with cultures of osteoblasts grown from bone chips. Cells are embedded in a dense meshwork of type I collagen fibers and mineral is observed in the extracellular matrix associated with collagen fibrils. Differentiating hypertrophic chondrocytes secrete large amounts of an 82-kD glycoprotein. The protein has been purified from conditioned medium and identified as ovotransferrin. It is transiently expressed during the in vitro differentiation of hypertrophic chondrocytes into osteoblast-like cells. In cultured hypertrophic chondrocytes treated with 500 nM retinoic acid, ovotransferrin is maximally expressed 3 d after retinoic acid addition, when the cartilage-bone-specific collagen shift occurs, and decays between the 5th and the 10th day, when cells have fully acquired the osteoblast-like phenotype. Similar results were obtained when retinoic acid was added to the culture at the 50 nM "physiological" concentration. Cells expressing ovotransferrin also coexpress ovotransferrin receptors. This suggests an autocrine mechanism in the control of chondrocyte differentiation to osteoblast-like cells.

Alkaline Phosphatase↗
PubMed1993

Type VI collagen expression is upregulated in the early events of chondrocyte differentiation.

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310-2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5-6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type VI collagen antibodies reveals that the early mRNA rise is paralleled by an increased secretion of type VI collagen in cell media. Induction of type VI collagen is not the consequence of trypsin treatment of dedifferentiated cells since exposure to the actin-disrupting drug cytochalasin or detachment of the cells by mechanical procedures has similar effects. In 13-day-old chicken embryo tibiae, where the full spectrum of the chondrogenic differentiation process is represented, expression of type VI collagen is restricted to the articular cartilage where chondrocytes developmental stage is comparable to stage I (high levels of type II collagen expression).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Expression of integrin receptors and their role in adhesion, spreading and migration of normal human melanocytes.

Integrin receptors of human melanocytes in vivo and of melanocytes isolated and cultured from in vitro reconstituted normal human epidermis were investigated. Melanocytes were studied by high-resolution immunocytochemistry of in situ epidermis and were found to expose only the integrin subunits alpha 3, alpha 6, alpha v and beta 1 on their plasma membrane surface. Instead, cultured normal melanocytes expressed alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha v beta 3, which were immunoprecipitated from both metabolically and surface-labeled cells. Beta 1 integrins were diffused on the adhesion surface, while alpha v beta 3 was clustered in focal contacts both in control cells and upon dendrite induction with phorbol 12-myristate 13-acetate (PMA). The functional roles of integrins were studied in vitro by cell adhesion, spreading and migration assays. The sum of the data indicated that, in normal human melanocytes: (i) adhesion to defined substrata is mainly mediated by specific beta 1 integrins; (ii) spreading is mainly modulated by alpha v beta 3; (iii) the beta 1 and beta 3 heterodimers cooperate in regulating migration. The in vitro expression of two integrins (alpha v beta 3 and alpha 5 beta 1) that are not exposed in situ, and their role in the spreading and migratory properties of melanocytes, strongly suggest that they are involved in regenerating a normally pigmented epidermis during wound healing by controlling melanocyte spreading and migration over a provisional matrix. Tumor promoters, such as PMA, selectively increased the expression of alpha 3 beta 1. We suggest that this integrin might be involved in melanocyte migration on the newly formed basement membrane during wound healing as well as in intercellular recognition of adjacent keratinocytes.

Cell Adhesion↗
PubMed1993

Alpha melanocyte stimulating hormone (alpha MSH) stimulates normal human melanocyte growth by binding to high-affinity receptors.

The combined action of cholera toxin (CT)-dependent activation of the adenylate cyclase signaling pathway, stimulation of protein kinase C, and activation of the tyrosine kinase activity of cell surface receptors and proto-oncogene products, have been shown to stimulate melanocyte proliferation. However, natural factors responsible for the optimal stimulation of normal human melanocyte growth, either isolated or co-cultured with keratinocytes, remain largely unknown. alpha MSH (alpha melanocyte stimulating hormone) has previously been shown to bind to murine and human melanoma cells and to stimulate their adenylate cyclase and tyrosinase activity. In contrast, very little is known about the presence and function of alpha MSH receptors in normal human melanocytes. We now report that alpha MSH: (i) binds to normal human melanocytes through a single class of high-affinity receptors; (ii) does not induce per se melanocytes to enter the S-phase of the cell cycle; (iii) does indeed stimulate melanocyte proliferation in a dose-dependent fashion; but its stimulatory effect requires bFGF and/or the activation of protein kinase C.

Adenylyl Cyclases↗
PubMed1992

Expression of epidermal growth factor receptor and transforming growth factor alpha in human larynx carcinoma.

Altered expression of growth factors and growth factor receptors is frequently described in human tumors and human tumor cell lines. This further supports the hypothesis that oncogenesis is due to the subversion of mitogen-responsive pathways. The aim of this study was to investigate the expression of epidermal growth factor receptor (EGFR) and transforming growth factor alpha (TGF alpha) in 13 larynx carcinomas and 2 carcinomas of the oral cavity. We found receptor overexpression in 7 out of 15 tumors at mRNA and/or protein level but low expression in the majority of the normal adjacent tissues. TGF alpha was expressed only in 1 case, but no tyrosine kinase activity of the receptor was detected by antiphosphotyrosine antibody.

Aged↗
PubMed1992

Heat-shock response in cultured chick embryo chondrocytes. Osteonectin is a secreted heat-shock protein.

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows 'in vitro' differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi-Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310-2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat-shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures. When we specifically investigated HSP species released into the culture medium, we observed a 43-45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM-40).

Animals↗