Sites Of Cell Proliferation Research Articles

A compendium of expression patterns of cholesterol biosynthetic enzymes in the mouse embryo

Cholesterol and its biosynthetic pathway intermediates and derivatives are required for many developmental processes including membrane biogenesis, transmembrane receptor signaling, steroid biogenesis, nuclear receptor activation, and posttranslational modification of hedgehog (Hh) proteins. To perform such multifaceted tasks depends on stringent regulation of expression of cholesterol biosynthetic enzymes (CBEs). We established for a whole organism, for the first time, the 3D expression pattern of all genes required for cholesterol biosynthesis (CBS), starting from acetyl-CoA and ending with cholesterol. This data was produced by high-throughput in situ hybridization on serial sections through the mouse fetus. The textually annotated image data were seamlessly integrated into the METscout and GenePaint public databases. This novel information helps in the understanding of why CBEs are expressed at particular locations within the fetus. For example, strong CBE expression is detected at sites of cell proliferation and also where cell growth increases membrane surface, such as in neurons sprouting axons and forming synapses. The CBE data also sheds light on the spatial relationship of cells and tissue that express sonic Hh (Shh) and produce cholesterol, respectively. We discovered that not all cells expressing Shh are capable of CBS. This finding suggests novel ways by which cholesterylation of Shh is regulated.

SUN2 accumulation contributes to vascular smooth muscle cell alterations induced by matrix rigidity

In the vascular system, arterial wall cell phenotype and fate are driven by the mechanical properties of their surrounding environment. Changes in the mechanical environment, such as age‐related extracellular matrix (ECM) stiffening, alter vascular cell phenotype and growth, leading to inflammation and vascular remodeling, which favor cardiovascular diseases. Despite recent advances, how mechanical cues regulate cell phenotype remains poorly understood. By combining proteomic and biophysical assays, we recently identified nuclear proteins whose expression or post translational modifications are regulated by matrix rigidity. Among the identified candidates is Sad1/UNC‐84 domain‐containing protein 2 (SUN2), which belongs to the linker of nucleoskeleton and cytoskeleton complex and enables mechanical force transmission from the cytoskeleton to the nucleus. We observed increased SUN2 expression in vascular smooth muscle cells (VSMCs) cultured on rigid matrix. Interestingly, we found that SUN2 depletion prevented the increase in cell proliferation in response to matrix rigidity. Using a mouse model of endothelial injury, we observed SUN2 accumulation in the neointima at sites of cell proliferation. To analyze VSMC contractile function we used atomic force microscopy (AFM). We found that SUN2 depletion decreased VSMC contractile response to AFM pulling forces. All together these results indicate that upregulation of SUN2 contributes to the increased proliferation and altered contractility of VSCM in response to matrix stiffening.

Sites of cell proliferation during scute morphogenesis in turtle and alligator are different from those of lepidosaurian scales

AbstractCell proliferation in forming shield scutes has been studied by immunofluorescence in embryos of turtle, alligator and snake after injection of 5‐bromo‐deoxy‐uridine. Hinge regions of scutes in alligator and turtle carapace derive from an initial waving and invagination of the epidermis that contains 5‐bromo‐deoxy‐uridine‐labelled cells. This suggests that down growth of the epidermis into the dermis is driven by local proliferation in addition to dermal anchorage and stabilization of hinge regions. Few keratinocytes migrate into suprabasal layers 1 day after injection of 5‐bromo‐deoxy‐uridine and keratinocytes reach the precorneous layer in about 5 days. Proliferating keratinocytes are randomly distributed in the outer scale surface of symmetric scutes but are more numerous in the outer scale surface of asymmetric or overlapped scutes indicating epidermal expansion. Higher localization of proliferating cells along hinge regions of embryonic turtle and alligator scutes is maintained in adult scutes where most growth occurs. In snake, skin proliferation becomes prevalent on the elongating outer side of the asymmetric scale. Comparison between proliferation sites in turtle–alligator–chick scales with lepidosaurian scales indicates that placodes are present only in turtle–alligator–chick scales. Conversely, scale primordia detected only using gene markers are found in most crocodilian and lepidosaurians embryonic skin.

Immunolocalization of Nestin in the lizard Podarcis muralis indicates up-regulation during the process of tail regeneration and epidermal differentiation

In order to study some of the proteins involved in tissue remodeling and regeneration, the stem cell marker and dynamic cytoskeletal protein nestin have been detected using immunocytochemistry in normal and regenerating tail of lizard. In tissues of the normal tail, nestin is only present in the alpha-layer of scales and in sparse cells of connective tissues. However, during tail regeneration, nestin is more commonly detected in various tissues, especially in the epidermis and regenerating scales, muscles, nerves and in the growing spinal cord. Sporadic nestin immunopositive cells are seen in the regenerating blastema and derived connective tissues. In the epidermis, nestin is expressed in the main sites of cell proliferation located in the apical alpha-keratinocytes of the forming scales, but disappears in beta-cells. In the regenerating muscles, nestin is seen in myoblasts and elongating myotubes, but disappears in mature muscles. Nestin appears in the growing nerves and in a few neurons of the regenerating spinal cord. It is present in the apical ependymal cells and in peripheral nerves and Schwann cells. The dynamic cytoskeletal protein nestin appears to be activated in the main sites of cell proliferation and cytoskeletal remodeling needed for re-epithelization and the differentiation of new scales, muscles and nervous tissue.

Cell-Cycle Control by Physiological Matrix Elasticity and In Vivo Tissue Stiffening

A number of adhesion-mediated signaling pathways and cell-cycle events have been identified that regulate cell proliferation, yet studies to date have been unable to determine which of these pathways control mitogenesis in response to physiologically relevant changes in tissue elasticity. In this report, we use hydrogel-based substrata matched to biological tissue stiffness to investigate the effects of matrix elasticity on the cell cycle. We find that physiological tissue stiffness acts as a cell-cycle inhibitor in mammary epithelial cells and vascular smooth muscle cells; subcellular analysis in these cells, mouse embryonic fibroblasts, and osteoblasts shows that cell-cycle control by matrix stiffness is widely conserved. Remarkably, most mitogenic events previously documented as extracellular matrix (ECM)/integrin-dependent proceed normally when matrix stiffness is altered in the range that controls mitogenesis. These include ERK activity, immediate-early gene expression, and cdk inhibitor expression. In contrast, FAK-dependent Rac activation, Rac-dependent cyclin D1 gene induction, and cyclin D1-dependent Rb phosphorylation are strongly inhibited at physiological tissue stiffness and rescued when the matrix is stiffened in vitro. Importantly, the combined use of atomic force microscopy and fluorescence imaging in mice shows that comparable increases in tissue stiffness occur at sites of cell proliferation in vivo. Matrix remodeling associated with pathogenesis is in itself a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1.

Dying to Get Out of the Crypt

Initially identified as a diffusible protein that guides axonal migration, netrin-1 has more recently been suggested to also function as a survival factor that inhibits the initiation of apoptotic pathways (see Fearon and Cho). Mazelin et al. investigated the possibility that, by acting as a dependence receptor, the netrin-1 receptor DCC (deleted in colorectal cancer) functions as a tumor suppressor. The authors used in situ hybridization and immunohistochemistry to show that, in the mouse colon and small intestine, netrin-1 mRNA and protein were localized to the bases of epithelial crypts (a site of cell proliferation) rather than villus tips (where cells die and are shed). DCC, however, was located throughout the epithelium. In transgenic mice that overexpressed gastrointestinal (GI) netrin-1, in which it was found throughout the epithelium, there was a 50% reduction in the number of apoptotic GI epithelial cells compared to that in wild-type mice, whereas cell proliferation and differentiation appeared unchanged. In contrast, mice lacking netrin-1 showed increased cell death. Moreover, 17% of the netrin-1-overexpressing mice (and none of the wild-type mice) developed GI adenomas. When netrin-1 overexpression was combined with a mutation in adenomatous polyposis coli (APC, which itself triggers the development of low-grade adenomas), 40% of the mice developed high-grade adenomas and half of those developed adenocarcinomas. Thus, the authors propose that a gradient of netrin-1 within intestinal crypts may normally lead to the death of cells at the villus tip and loss of DCC may promote colorectal cancer by eliminating the dependence of cell survival on the presence of netrin-1. L. Mazelin, A. Bernet, C. Bonod-Bidaud, L. Pays, S. Arnaud, C. Gespach, D. E. Bredesen, J. Y. Scoazec, P. Mehlen, Netrin-1 controls colorectal tumorigenesis by regulating apoptosis. Nature 431 , 80-84 (2004). [Online Journal] E. R. Fearon, K. R. Cho, Cell survival guide. Nature 431 , 35-36 (2004). [Online Journal]

Adrenocortical zonation and ACTH.

The clear morphological distinction between the cells of the different adrenocortical zones has attracted speculation and experiment to interpret their functions and the ways in which they are regulated. Considerable data have been produced in recent years that has benefited a fuller understanding of the processes of steroidogenesis and of cell proliferation at the molecular level. This now enables the reexamination of earlier concepts. It is evident that there is considerable species variation, and this article, dealing mainly with the rat, reaches conclusions that do not necessarily apply to other mammals. In the rat adrenal, however, the evidence suggests that the greatest differences between the functions of the zones are between the glomerulosa and the fasciculata. Here the sometimes all-or-nothing demarcation in their complement of components associated with steroidogenesis or with cell proliferation suggests a stark division of labor. In this model the fasciculata is the main engine of steroid hormone output and the glomerulosa is the site of cell proliferation, recruitment, and differentiation. Regulating these functions are angiotensin II and other paracrine components that modulate and maintain the glomerulosa, and ACTH, that maintains the fasciculata, and recruits new fasciculata cells by transformation of proliferating glomerulosa cells. Grafted onto this mostly vegetative function of the glomerulosa is CYP11B2, limited to just a fraction of the outer glomerulosa in rats on a normal laboratory diet and generating aldosterone (and 18-hydroxycorticosterone) from precursors whose origin is not, from the evidence summarized here, very clear, but may include the fasciculata, directly or indirectly. The biosynthesis of aldosterone in the rat certainly requires reinterpretation.

Characterization of a Serine Protease that Cleaves Pro-γ-Melanotropin at the Adrenal to Stimulate Growth

The adrenal gland requires stimuli from peptides derived from the ACTH precursor, pro-opiomelanocortin (POMC), to maintain its tonic state. Studies have proposed that a specific postsecretional cleavage of the nonmitogenic N-terminal 16 kDa fragment, also known as pro-γ-melanotropin (pro-γ-MSH), is required, releasing shorter fragments that promote adrenal growth. Here, we provide evidence for this hypothesis by the cloning and characterization of a serine protease that is upregulated during growth of the adrenal cortex. It is expressed exclusively in the outer adrenal cortex, the site of cell proliferation, and in the Y1 adrenal cell line. We also show that it is required for growth of Y1 cells, remains bound to the cell surface, and cleaves its substrate, pro-γ-MSH, at a specific bond.

Regulation of epidermal homeostasis through P2Y2 receptors.

1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Map kinase in the rat adrenal gland

The role of MAP Kinase (MAPK/ERK) in adrenal growth and steroidogenesis is unclear, though in other tissues it is known to act as an integrator of mitogenic signals originating from receptor tyrosine kinases and G-protein coupled receptors. Angiotensin II (AngII) is a major regulator of tissue differentiation and function in the adrenal, acting mainly through the AT1 receptor. Immunocytochemical and enzyme assay methods were used to study the distribution of MAPK and the action of AngII and associated antagonists saralasin and losartan(DuP753) in the rat adrenal gland. MAPK is localised in the zona glomerulosa (ZG) and the medulla, but absent from the zonae fasiculata and reticularis (ZF/ZR). Stimulation with AngII led to decreases in cytosolic and increases in nuclear MAPK activity, and its redistribution from the cytoplasm in unstimulated cells to its localisation around the nucleus, which was confirmed by immunocytochemistry. This translocation was inhibited in the presence of the AngII antagonist saralasin. Therefore, MAPK is located in the glomerulosa, where the AT1 receptor is localised and concerned with aldosterone biosynthesis, and in the medulla where MAPK activation results from AT2R activation. The results indicate the importance of the glomerulosa as the main site of cell proliferation in the adrenal cortex, and that MAPK may represent new signalling pathways related to zone function in the adrenal gland.

Adult neurogenesis in the telencephalon of a lizard: a [ 3H]thymidine autoradiographic and bromodeoxyuridine immunocytochemical study

A qualitative and quantitative evaluation of adult neurogenesis in the telencephalon of a lizard was conducted using [ 3H]thymidine autoradiography and immunocytochemical detection of 5′-bromodeoxyuridine (BrdU). The sites of cell proliferation, the fate, and the phenotype of cells born in adulthood were determined by short and long survival experiments (7 and 30 days). Adult specimens of the Moorish gecko, Tarentola mauritanica (Sauria, Gekkonidae), received subcutaneous injections of either BrdU or [ 3H]thymidine. Seven days after BrdU administration most labelled cells were in the walls (ventricular zone; VZ) of the lateral ventricles, particularly close to the sulcal zones. Labelled cells in the VZ of other ventricles were practically absent. Following 30 days, labelled cells outside the VZ were seen in most telencephalic regions: olfactory bulbs (OB), medial, dorsal, and lateral cortices (MC, DC, LC), anterior dorsal ventricular ridge (ADVR), nucleus sphericus (NS), and striatum (St). Electron microscopic examination of [ 3H]thymidine labelled cells found in these areas revealed their neuronal identity. No labelled neurons were detected in the dorsomedial cerebral cortex (DMC), septum (Sp), or elsewhere in the telencephalon. We conclude that neurogenesis occurs in most regions of the adult lizard telencephalon. The most intense neuronal production occurs in the MC. Other telencephalic regions, such as the OB, ADVR, and NS, also show a considerable rate of adult neurogenes. We infer from our results that the new neurons are born in the walls of the lateral ventricles and then migrate to their destinations. Furthermore, we report some evidence suggesting that neurons formed in adulthood in the olfactory bulbs are generated in the distant proliferative VZ. The functional significance of neurogenesis in the telencephalon of adult lizards in unclear, but it seems to be restricted to areas that have been implicated in olfactory plasticity, learning, and memory.

Validation of bromodeoxyuridine immunohistochemistry for localization of S-phase cells in decalcified tissues. A comparative study with tritiated thymidine autoradiography

Immunohistochemical detection of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdUrd), which is incorporated by S-phase cells, offers a convenient way of studying the proliferation kinetics of cells in normal skeletal tissues and in bone containing/derived tumours. To assess the validity of using this approach on decalcified, paraffin embedded tissues, the BrdUrd method was compared with tritiated thymidine (3H-TdR) autoradiography, using rat tibiae labelled with both 3H-TdR and BrdUrd, fixed in Carnoy's fluid and decalcified in EDTA, prior to routine paraffin embedding. The distribution of BrdUrd-labelled cells correlated with the sites of cell proliferation in the growing rat tibia. Independent studies with each method on paired serial sections of double-labelled tissue, showed a highly significant correlation (r = 0.81, p less than 0.0003) in the numbers of labelled cells seen in autoradiographs and immunostained sections from the proximal tibial growth plate. Combined BrdUrd immunohistochemistry and 3H-TdR autoradiography showed that the majority of labelled cells in cartilage, bone marrow, and fibrous perichondrium and periosteum had incorporated both labels. These results show that BrdUrd immunohistochemistry is a valid technique for the study of dividing cells in mineralized tissues after decalcification.

Etude autoradiographique de la proliferation et de la migration cellulaires au cours de la croissance larvaire de l'appareil visuel chez Aeshna cyanea Müll. (odonata : Aeshnidea)

Sites of cell proliferation in the ocular apparatus of Aeshna cyanea have been studied by an autoradiographic method ( 3H-thymidine). This technique was used to locate at the compound eye and optic lobe levels the zones of cellular multiplication and the migration of newly formed cells. In addition, the effect of the differential growth of the compound eye on the development of the optical lobe was studied. In the compound eye, two distinct levels of cell proliferation were revealed: the first one was the ocular budding zone itself, i.e. the ocular edge adjacent to the cephalic epidermis. A second mitotic focus, where the cells gather into ommatidial columns, was demonstrated. Thus, two closely related successive mitotic waves occur in the eye of this heterometabolous insect, whereas in holometabolous insects these two phases are much more distinct. Migration of ganglionic cells from the neuroblast masses, where they are formed, to the optical ganglions, where they differentiate, was also followed by this method. In addition, a differential growth of optic lobes was revealed: at the end of the nymphal life, the development was much greater in the posterior area of the optic lobes than in the anterior, although the numbers of cells produced in the neuroblast masses were, at the beginning, identical in both areas. This observation has been interpreted in the light of the experimental results.

The Cellular Basis for Colony Form inPodocoryne carnea

During the past 10 years considerable data have appeared indicating that the growth of hydroids is not, as was earlier believed, similar to the meristematic growth of plants, but rather that the sites of cell proliferation are removed in space from the sites of utilization, that cells migrate individually, actively as amoebocytes through the epidermis or pawively as epitheliocytes carried along in the hydroplasm, to the sites of utilization, and that considerable migration across the mesoglea occurs. A model of hydroid morphogenesis and morphostasis based on this new information provides a comprehensive picture, albeit one filled in with numerous assumptions, of the manner in which colony form is produced as a consequence of cell proclivities.

Natural variation in the S-phase of renewing epithelia of the pouchless opossum, Marmosa mitis.

ABSTRACTThe sites of cell proliferation and the duration of the S‐phases in epithelia (tongue, stomach, duodenum, jejunum, ileum and descending colon) of the pouchless opossum, Marmosamitis, have been studied following the injection of tritiated thymidine. the sites of cell proliferation in these epithelia are not significantly different than those reported for rodent tissues. On the other hand, measurements of the mean duration of DNA synthesis revealed great variability in this phase: tongue (12.8 hr), stomach (>14.0 hr), duodenum (8.5 hr), jejunum (8.6 hr), ileum (9.7 hr) and descending colon (11‐3 hr). In addition, the values obtained for the mean duration of t2 (G2+2/1M) are fairly constant among the various epithelia. It is concluded that the times obtained for the average duration of the S‐phases are longer and more variable in M. mitis than similar observations reported on renewing epithelia of eutherian mammals.

AUTORADIOGRAPHIC OBSERVATIONS ON THE LOCATION OF DNA-- AND RNA-- SYNTHESIZING CELLS IN RAT POPLITEAL LYMPH NODES.

Summary3H‐thymidine and 3H‐cytidine have been used to study the histological localization of DNA‐ and RNA‐synthesizing cells in popliteal lymph nodes from untreated, immunized and neonatally thymectomized rats. Active cell proliferation, as measured by 3H‐thymidine incorporation, was demonstrated in the medulla of unstimulated nodes from normal and from neonatally thymectomized rats. After an immunological stimulus, an early increase in medullary cell proliferation was associated with a similar but less intense reaction in the cortex. A partial dissociation between the sites of cell proliferation and the sites of most active RNA synthesis was noted. Cells with the highest apparent RNA‐synthetic rates were found predominantly in the cortex. Reticular cells, histiocytes, and plasma cells were found to retain labelled RNA longer than the other cells of the lymph nodes.The ability to achieve considerably more intense labelling in popliteal lymph node cells than in the rest of the lymphoid system by hind foot‐pad injections of 3H‐nucleosides was confirmed. However, attempts to use this finding for a study of lymph node cell migration were complicated by a failure to obtain adequate numbers of labelled cells after injection of 3H‐thymidine, and by the difficulty of finding a dose of 3H‐cytidine which labelled small lymphocytes in the popliteal nodes but not blast cells in other organs. It was possible, however, to demonstrate lymphocyte migration within 30 minutes of selective labelling of one popliteal node. Small numbers of migrant cells were found in other lymph nodes, spleen, thymus, and ileal submucosa.