A dual effector theory of growth-hormone action

Growth hormone increases tissue formation by acting both directly and indirectly on target cells. The direct action promotes the differentiation of precursor cells; this has been demonstrated for two mesenchymal cell types. Insulin-like growth factor I (IGF-I) is not able to substitute for growth hormone in promoting this differentiation, but it is proposed that its mitogenic action selectively promotes cell multiplication in young differentiated clones. As tissue growth results from both the creation of new differentiated cells and their subsequent clonal expansion, both effectors increase tissue growth, but by different means.     

Müllerian inhibitory substance induces growth of rat preantral ovarian follicles

Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is best known as the hormone that regulates the regression of the Müllerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Müllerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis.     

Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf

Coordination of cell proliferation and differentiation is crucial for tissue formation, repair and regeneration. Some tissues, such as skin and blood, depend on differentiation of a pluripotent stem cell population, whereas others depend on the division of differentiated cells. In development and in the hair follicle, pigmented melanocytes are derived from undifferentiated precursor cells or stem cells. However, differentiated melanocytes may also have proliferative capacity in animals, and the potential for differentiated melanocyte cell division in development and regeneration remains largely unexplored. Here, we use time-lapse imaging of the developing zebrafish to show that while most melanocytes arise from undifferentiated precursor cells, an unexpected subpopulation of differentiated melanocytes arises by cell division. Depletion of the overall melanocyte population triggers a regeneration phase in which differentiated melanocyte division is significantly enhanced, particularly in young differentiated melanocytes. Additionally, we find reduced levels of Mitf activity using an mitfa temperature-sensitive line results in a dramatic increase in differentiated melanocyte cell division. This supports models that in addition to promoting differentiation, Mitf also promotes withdrawal from the cell cycle. We suggest differentiated cell division is relevant to melanoma progression because the human melanoma mutation MITF(4T)(Δ)(2B) promotes increased and serial differentiated melanocyte division in zebrafish. These results reveal a novel pathway of differentiated melanocyte division in vivo, and that Mitf activity is essential for maintaining cell cycle arrest in differentiated melanocytes.     

VEGF-R2 and neuropilin-1 are involved in VEGF-A-induced differentiation of human bone marrow progenitor cells

Tumor growth and metastasis require the generation of new blood vessels, a process known as neo-angiogenesis. Recent studies have indicated that early tumor vascularization is characterized by the differentiation and mobilization of human bone marrow cells. Vascular endothelial growth factor-A (VEGF-A) is one of the growth factors, which enhances their differentiation into endothelial cells, but little is known about the implication of the VEGF-receptor tyrosine kinases and about the implication of the VEGF-R co-receptor, neuropilin-1, in this process. In this context, the identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells was investigated in order to define the pharmaceutical targets involved in tissue vascularization associated with this process. For this purpose, an in vitro model of differentiation of human bone marrow AC133+ (BM-AC133+) cells into vascular precursors was used. In this work, we have demonstrated for the first time that the effect of VEGF-A on BM-AC133+ cells relies on an early action of VEGF-A on the expression of its tyrosine kinase receptors followed by an activation of a VEGF-R2/neuropilin-1-dependent signaling pathway. This signaling promotes the differentiation of BM-AC133+ cells into endothelial precursor cells, followed by the proliferation of these differentiated cells. Altogether, these results strongly suggest that VEGF inhibitors, acting at the level of VEGF-R2 and/or neuropilin-1, by inhibiting differentiation and proliferation of these cells, could be potentially active compounds to prevent progenitor cells to be involved in tumor angiogenesis leading to tumor growth.     

Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin

Summary During postembryonic development of insects, molting cycles affect epidermal cells with alternate periods of proliferation and differentiation. Cells of the cell line established from imaginal discs of the Indian meal moth (IAL-PID2) differentiate under the action of the molting hormone, 20-hydroxyecdysone, in a manner that is meaningful in terms of the development of the tissue from which they were derived. In particular, the hormone caused an accumulation of the cells in the G2 phase of their cycle and induced the formation of epithelial-like aggregates and the synthesis of specific proteoglycans. Recent discovery of members of the insulin superfamily in insects and the role of growth factors played by this family of molecules in vertebrates led us to check for their potential effects on IAL-PID2 cell cycle regulation. On the one hand, our results showed that insulin was involved in partial resumption of the cell cycle after an arrest caused by serum deprivation, but that other growth factors present in fetal calf serum were needed for full completion of mitosis. On the other hand, the cytostatic effect of 20-hydroxyecdysone was reversible, and, prior exposure of the cells to the hormone allowed the cells to complete one cell cycle in serum-free medium. These results suggest that the production of autocrine growth factors induced by ecdysteroids could circumvent the absence of serum. This cell culture model provides potential for further study of interactions between ecdysteroids and growth factor homologs during differentiation of insect epidermal cells.     

Notch is the key factor in the process of fetal liver stem/progenitor cells differentiation into hepatocytes

Cell transplantation is efficient method to therapy end-stage liver disease (ESLD). How to punctually induce stem cell differentiation into hepatocyte is still a challenge. Notch plays important roles in embryonic development and cell differentiation. However, during the differentiation process from fetal liver stem/progenitor cells (FLSPCs) to mature hepatocytes, the contribution of Notch, especially which Notch receptor is primarily responsible, is unknown. First, specific Notch receptor responsible for FLSPCs differentiation was identified. On both tissue level and cell level, we found that Notch3 was the only receptor greater expressed in liver tissue at embryonic day (ED) 14 and FLSPCs, compared with the adult liver and BRL cells, respectively. Second, morphological phenotypic and functional aspects were analyzed to evaluate whether Notch inhibition by GSIs (γ-secretase inhibitors, inhibitor of Notch) promotes the differentiation of FLSPCs into hepatocytes. Results showed that N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) as GSIs was able to induce FLSPCs differentiation into hepatocytes. The differentiated FLSPCs showed similar morphology to mature hepatocytes, expressed hepatic markers indicative of a mature developmental stage, and displayed similar functionality to mature hepatocytes. The differentiation efficiency by GSIs was similar to that by hepatocyte growth factor (HGF) induction. More specifically, as the differentiation of FLSPCs progressed towards hepatocytes, the expression of Notch3 was gradually down-regulated, consistent with the down-regulation of other stem cell markers. These findings imply that Notch3 may not only be a regulator of FLSPCs differentiation into hepatocytes, but also be a potential marker of FLSPCs.     

CINCINNATA controls both cell differentiation and growth in petal lobes and leaves of Antirrhinum

To understand how differentiation and growth may be coordinated during development, we have studied the action of the CINCINNATA (CIN) gene of Antirrhinum. We show that in addition to affecting leaf lamina growth, CIN affects epidermal cell differentiation and growth of petal lobes. Strong alleles of cin give smaller petal lobes with flat instead of conical cells, correlating with lobe-specific expression of CIN in the wild type. Moreover, conical cells at the leaf margins are replaced by flatter cells, indicating that CIN has a role in cell differentiation of leaves as well as petals. A weak semidominant cin allele affects cell types mainly in the petal but does not affect leaf development, indicating these two effects can be separated. Expression of CIN correlates with expression of cell division markers, suggesting that CIN may influence petal growth, directly or indirectly, through effects on cell proliferation. For both leaves and petals, CIN affects growth and differentiation of the more distal and broadly extended domains (leaf lamina and petal lobe). However, while CIN promotes growth in petals, it promotes growth arrest in leaves, possibly because of different patterns of growth control in these systems.     

Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O- sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.      

Growth hormone receptor expression in the nucleus and cytoplasm of normal and neoplastic cells

 Growth hormone (GH) exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific receptors which trigger a phosphorylation cascade, resulting in the modulation of numerous signalling pathways dictating gene expression. A panel of five monoclonal antibodies was used in mapping the presence and somatic distribution of the GH receptor by immunohistochemistry in normal and neoplastic tissues and cultured cells of human, rat and rabbit origin. A wide distribution of the receptor was observed in many cell types. Not all cells expressing cytoplasmic GH receptors displayed nuclear immunoreactivity. In general, the relative proportion of positive cells and intensity of staining was higher in neoplastic cells than in normal tissue cells. Immunoreactivity showed subcellular localisation of the GH receptor in cell membranes and was predominantly cytoplasmic, but strong nuclear immunoreaction was also apparent in many instances. Intense immunoreactivity was also observed in the cellular Golgi area of established cell lines and cultured tissue-derived cells in exponential growth phase, indicating cells are capable of GH receptor synthesis. The presence of intracellular GH receptor, previously documented in normal tissues of mostly animal origin, is the result of endoplasmic reticulum and Golgi localisation. Heterogeneity of immunoreactivity was found in normal and neoplastic tissue with a variable range of positive cells. The nuclear localisation of immunoreactivity is the result of nuclear GH receptor/binding protein, identically to the cytosolic and plasma GH-binding protein, using a panel of five monoclonal antibodies against the GH receptor extracellular region. The expression of GH receptors, not only on small proliferating tumour cells such as lymphocytes, but also on well differentiated cells including keratinocytes, suggests that GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion, differentiation and maintenance. Accepted: 4 July 1997     

Role of the extracellular matrix in morphogenesis

The extracellular matrix is a complex, dynamic and critical component of all tissues. It functions as a scaffold for tissue morphogenesis, provides cues for cell proliferation and differentiation, promotes the maintenance of differentiated tissues and enhances the repair response after injury. Various amounts and types of collagens, adhesion molecules, proteoglycans, growth factors and cytokines or chemokines are present in the tissue- and temporal-specific extracellular matrices. Tissue morphogenesis is mediated by multiple extracellular matrix components and by multiple active sites on some of these components. Biologically active extracellular matrix components may have use in tissue repair, regeneration and engineering, and in programming stem cells for tissue replacement.     

Appearance of functional insulin receptors during the differentiation of embryonal carcinoma cells

The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cells were investigated for both the embryo carcinoma (EC) and retinoic acid-induced differentiated derivatives of this cell line. Whereas the EC cell was found to have very few, if any, receptors and showed no demonstrable dependence on insulin for growth, the differentiated derivative cell expressed a large number of insulin receptors and, when challenged with the hormone, showed stimulation of both DNA synthesis and cell division. The same data were obtained for five independent PC13 clones. These results, coupled with previous observations, lend weight to the suggestion that the appearance of specific receptors for growth regulatory substances may be a manifestation of a general change in growth-regulatory mechanisms accompanying EC cell differentiation and loss of malignancy.     

Cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in the neural differentiation of mesenchymal stem cells in vitro

Previously, we found that suppressing phosphatidylcholine-specific phospholipase C could induce neuronal differentiation of rat mesenchymal stem cells in the absence of serum and fibroblast growth factor. It is well known that basic fibroblast growth factor plays an important role in mesenchymal stem cell neuronal differentiation. In this study, our purpose was to understand the cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in controlling mesenchymal stem cell neuronal differentiation. Our results showed that suppressing phosphatidylcholine-specific phospholipase C in the presence of basic fibroblast growth factor could induce cell neuronal differentiation and the viability of the differentiated cells was obviously increased. Furthermore, we found that the resting membrane potential of the differentiated cells gradually decreased, but the mitochondrial membrane potential rose with increasing treatment time and these characteristics were similar to cultured neurons from mouse embryo forebrains. To determine the possible mechanism by which this combination controls cell neuronal differentiation, we measured changes in the mitochondrial membrane potential and in the levels of reactive oxygen species. The results showed that both the mitochondrial membrane potential and reactive oxygen species levels decreased when basic fibroblast growth factor was added. The data suggested that lower phosphatidylcholine-specific phospholipase C activity was required for mesenchymal stem cell neuronal differentiation and basic fibroblast growth factor was necessary for maintaining the neuronal differentiation state. Moreover, basic fibroblast growth factor could contribute to rescuing the differentiated cells from death through decreasing overly high mitochondrial membrane potentials and reactive oxygen species levels.     

Properties of growth hormone receptors in relation to the adipose conversion of 3T3 cells

Cultured preadipose 3T3 cells undergo a process of differentiation in which they convert to adipose cells. Growth hormone promotes this conversion. Since 3T3 sublines vary in their susceptibility to adipose conversion, it was of interest to examine the properties of the growth hormone receptors in relation to that susceptibility. It was found that preadipose 3T3-F442A cells, which are able to convert to adipose cells with high frequency, are able to bind about 10(4) growth hormone molecules per cell with Kd approximately 10(-9) M. After adipose conversion, no appreciable change in hormone binding was detected. The binding of growth hormone to 3T3-C2 cells (a line virtually insusceptible to adipose conversion) was indistinguishable from that to 3T3-F442A cells. Internalization and degradation of the hormone were also similar in the two cell lines. Susceptibility to adipose conversion is therefore not determined by the relative ability of the cells to bind or degrade the hormone, but must instead depend on some response, as yet unidentified, that follows binding of the hormone.     

Unifying heuristic model of transmembrane co-ordinate control for cell growth and cell movement

An unifying dualistic system is proposed as control transmembrane mediator for both cell growth and cell movement. In this system, sparsely distributed membrane units containing allosteric protomers act through long-range co-operative phase transitions, to store or release a growth primary stereospecific initiator at the inner membrane surface. Other mosaic distributed membrane units contain movement modulation promoters; their mobility is controlled by a mechanism based on the microfilament-microtubule assemblage hypothesis. The movement inhibition is graded with the spreading of cell-cell contacts, while the growth control is an all-or-none effect depending on critical level of environmental factors. It is hypothesized that the growth controlling transmembrane effect is reversible in most of normal cultured cells and in mature cells having ability of proliferate, e.g. hepatocytes, but that it permanently promotes growth in the embryonic and tumor cells and is lacking in some highly differentiated cell types. This both mechanistic and metabolic transmembrane co-ordinate control allows to rationalize in an uniform model numerous different cell behavior phenomena, in particular surface modulation events, overlapping, contact inhibition of growth and movement, G,, S phase conversion, embryological development, differentiation in mature organisms, and metastasis. Its plausibility and implications are discussed.     

Somatomedin C/insulin-like growth factor 1: an intratesticular differentiative factor of Leydig cells?

By using immature porcine Sertoli cells cultured in serum-free defined medium, we report that medium conditioned by Sertoli cells contained immunoreactive somatomedin C/insulin-like growth factor 1 (SmC/IGF1) measured following acidic gel filtration. The release of this immunoreactive SmC/IGF1 was slightly increased following Sertoli cell treatment with fibroblast growth factor but not with follicle-stimulating hormone or growth hormone. On the other hand, human biosynthetic SmC/IGF1 exerts a potent stimulatory effect on Leydig cell differentiated functions such as LH/hCG-binding (greater than 4-fold) and hCG-stimulated testosterone secretion (greater than 15-fold). This effect was dose and time dependent and the maximal increase of Leydig cell function was observed following 48 h treatment with 50 ng/ml SmC/IGF1. The steroidogenic action of the peptide was not related to Leydig cell growth since both cell number and 3H-thymidine incorporation into DNA were not or slightly (approximately equal to 1.5-fold) increased in the optimal conditions with SmC/IGF1 treatment (100 ng/ml for 48 h). Moreover, the concomitant treatment of Leydig cells by both arabinoside C (10(-5) M), a DNA synthesis inhibitor, and SmC/IGF1 did not modify the stimulating effect of the peptide on LH/hCG-binding and hCG-stimulated testosterone production. Taken together, the present findings support the concept that Sertoli cell derived SmC/IGF1 could be a potent regulator of Leydig cell differentiated functions.     

The arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems

ATHB-8, -9, -14, -15, and IFL1/REV are members of a small homeodomain-leucine zipper family whose genes are characterized by expression in the vascular tissue. ATHB-8, a gene positively regulated by auxin (Baima et al., 1995), is considered an early marker of the procambial cells and of the cambium during vascular regeneration after wounding. Here, we demonstrate that although the formation of the vascular system is not affected in athb8 mutants, ectopic expression of ATHB-8 in Arabidopsis plants increased the production of xylem tissue. In particular, a careful anatomical analysis of the transgenic plants indicated that the overexpression of ATHB-8 promotes vascular cell differentiation. First, the procambial cells differentiated precociously into primary xylem. In addition, interfascicular cells also differentiated precociously into fibers. Finally, the transition to secondary growth, mainly producing xylem, was anticipated in transgenic inflorescence stems compared with controls. The stimulation of primary and secondary vascular cell differentiation resulted in complex modifications of the growth and development of the ATHB-8 transgenic plants. Taken together, these results are consistent with the hypothesis that ATHB-8 is a positive regulator of proliferation and differentiation, and participates in a positive feedback loop in which auxin signaling induces the expression of ATHB-8, which in turn positively modulates the activity of procambial and cambial cells to differentiate.     

Regulation of new fat cell formation

The formation of new adipocytes occurs either at the stage of multiplication or differentiation or both. It seems possible that the formation of new fat cells is dependent on the average cell weight in a given adipose tissue depot, but there may also be other regional, local regulatory factors. Multiplication of fat cells has been suggested to be stimulated by 17-beta-oestradiol while the differentiation of adipocytes is stimulated by growth hormone, glucocorticoids, insulin, insulin-like growth factor and female sex hormones. There are, probably, other factors acting in circulation or locally. The factors promoting growth of new fat cells with overfeeding are at present unknown. Some hypothetical possibilities are discussed.