Morphogenesis of the peri-implant mucosa: an experimental study in dogs

PURPOSE: The objective of the present experiment was to study the morphogenesis of the mucosal attachment to implants made of c.p. titanium. MATERIAL AND METHODS: All mandibular premolars were extracted in 20 Labrador dogs. After a healing period of 3 months, four implants (ITI Dental Implant System) were placed in the right and left sides of the mandible. A non-submerged implant installation technique was used and the mucosal tissues were secured to the conical marginal portion of the implants with interrupted sutures. The sutures were removed after 2 weeks and a plaque control program including daily cleaning of the remaining teeth and the implants was initiated. The animals were sacrificed and biopsies were obtained at various intervals to provide healing periods extending from Day 0 (2 h) to 12 weeks. The mandibles were removed and placed in the fixative. The implant sites were dissected using a diamond saw and processed for histological analysis. RESULTS: Large numbers of neutrophils infiltrated and degraded the coagulum that occupied the compartment between the mucosa and the implant during the initial phase of healing. At 2 weeks after surgery, fibroblasts were the dominating cell population in the connective tissue interface but at 4 weeks the density of fibroblasts had decreased. Furthermore, the first signs of epithelial proliferation were observed in specimens representing 1-2 weeks of healing and a mature barrier epithelium occurred after 6-8 weeks of healing. The collagen fibers of the mucosa were organized after 4-6 weeks of healing. CONCLUSION: It is suggested that the soft-tissue attachment to implants placed using a non-submerged installation procedure is properly established after several weeks following surgery.

Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.

LMW-E MEDIATES MAMMARY TUMORIGENESIS BY DEREGULATING ACINAR MORPHOGENESIS & GENERATING CANCER STEM CELLS

Cyclin E is the regulatory subunit of the cyclin E/CDK2 complex that mediates the G1-S phase transition. N-terminal cleavage of cyclin E by elastase in breast cancer generates two low molecular weight (LMW) isoforms that exhibit both enhanced kinase activity and resistance to p21 and p27 inhibition compared to fulllength cyclin E. Clinically, approximately 27% of breast cancer patients overexpress LMW-E and associate with poor survival. Therefore, we hypothesize that LMW-E disrupts normal mammary acinar morphogenesis and serves as the initial route into breast tumor development. We first demonstrate that LMW-E overexpression in non-tumorigenic hMECs is sufficient to induce tumor formation in athymic mice significantly more than overexpression of full-length cyclin E and requires CDK2- associated kinase activity. Further in vivo passaging of these tumors augments LMW-E expression and tumorigenic potential. When subjected to acinar morphogenesis in vitro, LMW-E mediates significant morphological disruption by generating hyperproliferative and multi-acinar complexes. Proteomic analysis of patient tissues and tumor cells with high LMW-E expression reveals that the activation of the b-Raf-ERK1/2-mTOR pathway in concert with high LMW-E expression predicts poor patient survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (b-raf inhibitor) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing the G1/S cell cycle arrest. In addition, the LMW-E-expressing tumor cells exhibit phenotypes characteristic of the EMT and enhanced cellular invasiveness. These tumor cells also enrich for cells with CSC phenotypes such as increased CD44hi/CD24lo population, enhanced mammosphere formation, and upregulation of ALDH expression and enzymatic activity. Furthermore, the CD44hi/CD24lo population also shows positive correlation with LMW-E expression in both the tumor cell line model and breast cancer patient samples (p<0.0001 & p=0.0435, respectively). Combination treatment using doxorubicin and salinomycin demonstrates synergistic cytotoxic effects in cells with LMW-E expression but not in those with full-length cyclin E expression. Finally, ProtoArray microarray identifies Hbo1 as a novel substrate of the cyclin E/CDK2 complex and its overexpression results in enrichment for CSCs. Collectively, these data emphasize the strong oncogenic potential of LMW-E in mammary tumorigenesis and suggest possible therapeutic strategies to treat breast cancer patients with high LMW-E expression.

The transcriptional repressor CDP (Cutl1) is essential for epithelial cell differentiation of the lung and the hair follicle

The mammalian Cutl1 gene codes for the CCAAT displacement protein (CDP), which has been implicated as a transcriptional repressor in diverse processes such as terminal differentiation, cell cycle progression, and the control of nuclear matrix attachment regions. To investigate the in vivo function of Cutl1, we have replaced the C-terminal Cut repeat 3 and homeodomain exons with an in-frame lacZ gene by targeted mutagenesis in the mouse. The CDP-lacZ fusion protein is retained in the cytoplasm and fails to repress gene transcription, indicating that the Cutl1(lacZ) allele corresponds to a null mutation. Cutl1 mutant mice on inbred genetic backgrounds are born at Mendelian frequency, but die shortly after birth because of retarded differentiation of the lung epithelia, which indicates an essential role of CDP in lung maturation. A less pronounced delay in lung development allows Cutl1 mutant mice on an outbred background to survive beyond birth. These mice are growth-retarded and develop an abnormal pelage because of disrupted hair follicle morphogenesis. The inner root sheath (IRS) is reduced, and the transcription of Sonic hedgehog and IRS-specific genes is deregulated in Cutl1 mutant hair follicles, consistent with the specific expression of Cutl1 in the progenitors and cell lineages of the IRS. These data implicate CDP in cell-lineage specification during hair follicle morphogenesis, which resembles the role of the related Cut protein in specifying cell fates during Drosophila development.

Reciprocal interactions between β1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology

Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of β1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4–2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a three-dimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of β1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogen-activated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant down-regulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Cross-modulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and β1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of β1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be “normalized” by manipulating either pathway.

Overexpression of a Kinase-deficient Transforming Growth Factor-β Type II Receptor in Mouse Mammary Stroma Results in Increased Epithelial Branching

Members of the transforming growth factor-β (TGF-β) superfamily signal through heteromeric type I and type II serine/threonine kinase receptors. Transgenic mice that overexpress a dominant-negative mutation of the TGF-β type II receptor (DNIIR) under the control of a metallothionein-derived promoter (MT-DNIIR) were used to determine the role of endogenous TGF-βs in the developing mammary gland. The expression of the dominant-negative receptor was induced with zinc and was primarily localized to the stroma underlying the ductal epithelium in the mammary glands of virgin transgenic mice from two separate mouse lines. In MT-DNIIR virgin females treated with zinc, there was an increase in lateral branching of the ductal epithelium. We tested the hypothesis that expression of the dominant-negative receptor may alter expression of genes that are expressed in the stroma and regulated by TGF-βs, potentially resulting in the increased lateral branching seen in the MT-DNIIR mammary glands. The expression of hepatocyte growth factor mRNA was increased in mammary glands from transgenic animals relative to the wild-type controls, suggesting that this factor may play a role in TGF-β-mediated regulation of lateral branching. Loss of responsiveness to TGF-βs in the mammary stroma resulted in increased branching in mammary epithelium, suggesting that TGF-βs play an important role in the stromal–epithelial interactions required for branching morphogenesis.

Genetic programs of epithelial cell plasticity directed by transforming growth factor-β

Epithelial–mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-β (TGF-β) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-β-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-β within 4 hours after treatment. TGF-β-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell–matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-β. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

Coordinated morphogenesis of epithelia during development of the Caenorhabditis elegans uterine-vulval connection.

Development of the nematode egg-laying system requires the formation of a connection between the uterine lumen and the developing vulval lumen, thus allowing a passage for eggs and sperm. This relatively simple process serves as a model for certain aspects of organogenesis. Such a connection demands that cells in both tissues become specialized to participate in the connection, and that the specialized cells are brought in register. A single cell, the anchor cell, acts to induce and to organize specialization of the epidermal and uterine epithelia, and registrates these tissues. The inductions act via evolutionarily conserved intercellular signaling pathways. The anchor cell induces the vulva from ventral epithelial cells via the LIN-3 growth factor and LET-23 transmembrane tyrosine kinase. It then induces surrounding uterine intermediate precursors via the receptor LIN-12, a founding member of the Notch family of receptors. Both signaling pathways are used multiple times during development of Caenorhabditis elegans. The outcome of the signaling is context-dependent. Both inductions are reciprocated. After the anchor cell has induced the vulva, it stretches toward the induced vulval cells. After the anchor cell has induced specialized uterine intermediate precursor cells, it fuses with a subset of their progeny.

Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development.

At least two kidney epithelial cell lines, the Madin-Darby canine kidney (MDCK) and the murine inner medullary collecting duct line mIMCD-3, can be induced to form branching tubular structures when cultured with hepatocyte growth factor (HGF) plus serum in collagen I gels. In our studies, whereas MDCK cells remained unable to form tubules in the presence of serum alone, mIMCD-3 cells formed impressive branching tubular structures with apparent lumens, suggesting the existence of specific factors in serum that are tubulogenic for mIMCD-3 cells but not for MDCK cells. Since normal serum does not contain enough HGF to induce tubulogenesis, these factors appeared to be substances other than HGF. This was also suggested by another observation: when MDCK cells or mIMCD-3 cells were cocultured under serum-free conditions with the embryonic kidney, both cell types formed branching tubular structures similar to those induced by HGF; however, only in the case of MDCK cells could this be inhibited by neutralizing antibodies against HGF. Thus, the embryonic kidney produces growth factors other than HGF capable of inducing tubule formation in the mIMCD-3 cells. Of a number of growth factors examined, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to be tubulogenic for mIMCD-3 cells. Whereas only HGF was a potent tubulogenic factor for MDCK cells, HGF, TGF-alpha, and EGF were potent tubulogenic factors for mIMCD-3 cells. Nevertheless, there were marked differences in the capacity of these tubulogenic factors to induce tubulation as well as branching events in those tubules that did form (HGF >> TGF-alpha > EGF). Thus, at least three different growth factors can induce tubulogenesis and branching in a specific epithelial cell in vitro (though to different degrees), and different epithelial cells that are capable of forming branching tubular structures demonstrate vastly different responses to tubulogenic growth factors. The results are discussed in the context of branching morphogenesis during epithelial tissue development.

Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells.

Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligand-induced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.

Localization of neogenin protein during morphogenesis in the mouse embryo

Neogenin, a close relative of the axon guidance receptor DCC, has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule families. Recent studies have begun to uncover a role for Neogenin in organogenesis. Here we examine the localization of Neogenin protein in the developing mouse embryo (embryonic day 14.5) when organogenesis is progressing rapidly. We observe that Neogenin protein is restricted to distinct tissue layers within a given organ. In some embryonic epithelia such as the gut and pancreas, Neogenin protein is predominantly polarized to the basal surfaces of the epithelial cells. In contrast, Neogenin is restricted to mesenchymal cells within the lung and kidney. Neogenin is also seen in differentiating skeletal muscle and condensing cartilage throughout the embryo, and in the trigeminal and dorsal root ganglia of the peripheral nervous system. This study supports the emerging role for Neogenin as a key receptor in the establishment of the morphological architecture in many developing organ systems.

Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.

Desquamation Is A Novel Phenomenon For Collective Prostate Epithelial Cell Deletion After Castration.

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

Analysis Of The Contribution Of Immunologically-detectable Her2, Steroid Receptors And Of The Triple-negative" Tumor Status To Disease-free And Overall Survival Of Women With Epithelial Ovarian Cancer."

We assessed associations between steroid receptors including: estrogen-alpha, estrogen-beta, androgen receptor, progesterone receptor, the HER2 status and triple-negative epithelial ovarian cancer (ERα-/PR-/HER2-; TNEOC) status and survival in women with epithelial ovarian cancer. The study included 152 women with primary epithelial ovarian cancer. The status of steroid receptor and HER2 was determined by immunohistochemistry. Disease-free and overall survival were calculated and compared with steroid receptor and HER2 status as well as clinicopathological features using the Cox Proportional Hazards model. A mean follow-up period of 43.6 months (interquartile range=41.4 months) was achieved where 44% of patients had serous tumor, followed by mucinous (23%), endometrioid (9%), mixed (9%), undifferentiated (8.5%) and clear cell tumors (5.3%). ER-alpha staining was associated with grade II-III tumors. Progesterone receptor staining was positively associated with a Body Mass Index≥25. Androgen receptor positivity was higher in serous tumors. In stand-alone analysis of receptor contribution to survival, estrogen-alpha positivity was associated with greater disease-free survival. However, there was no significant association between steroid receptor expression, HER2 status, or TNEOC status, and overall survival. Although estrogen-alpha, androgen receptor, progesterone receptor and the HER2 status were associated with key clinical features of the women and pathological characteristics of the tumors, these associations were not implicated in survival. Interestingly, women with TNEOC seem to fare the same way as their counterparts with non-TNEOC.

Key Participants Of The Tumor Microenvironment Of The Prostate: An Approach Of The Structural Dynamic Of Cellular Elements And Extracellular Matrix Components During Epithelial-stromal Transition.

Cancer is a multistep process that begins with the transformation of normal epithelial cells and continues with tumor growth, stromal invasion and metastasis. The remodeling of the peritumoral environment is decisive for the onset of tumor invasiveness. This event is dependent on epithelial-stromal interactions, degradation of extracellular matrix components and reorganization of fibrillar components. Our research group has studied in a new proposed rodent model the participation of cellular and molecular components in the prostate microenvironment that contributes to cancer progression. Our group adopted the gerbil Meriones unguiculatus as an alternative experimental model for prostate cancer study. This model has presented significant responses to hormonal treatments and to development of spontaneous and induced neoplasias. The data obtained indicate reorganization of type I collagen fibers and reticular fibers, synthesis of new components such as tenascin and proteoglycans, degradation of basement membrane components and elastic fibers and increased expression of metalloproteinases. Fibroblasts that border the region, apparently participate in the stromal reaction. The roles of each of these events, as well as some signaling molecules, participants of neoplastic progression and factors that promote genetic reprogramming during epithelial-stromal transition are also discussed.