Role of insulin-like growth factors and myogenin in the altered program of proliferation and differentiation in the NFB4 mutant muscle cell line.

In the present study we used the mutant muscle cell line NFB4 to study the balance between proliferation and myogenic differentiation. We show that removal of serum, which induced the parental C2C12 cells to withdraw from the cell cycle and differentiate, had little effect on NFB4 cells. Gene products characteristic of the proliferation state, such as c-Jun, continued to accumulate in the mutant cells in low serum, whereas those involved in differentiation, like myogenin, insulin-like growth factor II (IGF-II), and IGF-binding protein 5 (IGFBP-5) were undetectable. Moreover, NFB4 cells displayed a unique pattern of tyrosine phosphorylated proteins, especially in low serum, suggesting that the signal transduction pathway(s) that controls differentiation is not properly regulated in these cells. Treatment of NFB4 cells with exogenous IGF-I or IGF-II at concentrations shown to promote myogenic differentiation in wild-type cells resulted in activation of myogenin but not MyoD gene expression, secretion of IG-FBP-5, changes in tyrosine phosphorylation, and enhanced myogenic differentiation. Similarly, transfection of myogenin expression constructs also enhanced differentiation and resulted in activation of IGF-II expression, showing that myogenin and IGF-II cross-activate each other's expression. However, in both cases, the expression of Jun mRNA remained elevated, suggesting that IGFs and myogenin cannot overcome all aspects of the block to differentiation in NFB4 cells.

Identification and characterization of an immunophilin expressed during the clonal expansion phase of adipocyte differentiation.

Mouse 3T3-L1 cells differentiate into fat-laden adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the beta and delta members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the alpha form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP alpha, and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP beta and C/EBP delta, is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.

Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex.

Failure of programmed cell death and differentiation as causes of tumors: some simple mathematical models.

Most models of tumorigenesis assume that the tumor grows by increased cell division. In these models, it is generally supposed that daughter cells behave as do their parents, and cell numbers have clear potential for exponential growth. We have constructed simple mathematical models of tumorigenesis through failure of programmed cell death (PCD) or differentiation. These models do not assume that descendant cells behave as their parents do. The models predict that exponential growth in cell numbers does sometimes occur, usually when stem cells fail to die or differentiate. At other times, exponential growth does not occur: instead, the number of cells in the population reaches a new, higher equilibrium. This behavior is predicted when fully differentiated cells fail to undergo PCD. When cells of intermediate differentiation fail to die or to differentiate further, the values of growth parameters determine whether growth is exponential or leads to a new equilibrium. The predictions of the model are sensitive to small differences in growth parameters. Failure of PCD and differentiation, leading to a new equilibrium number of cells, may explain many aspects of tumor behavior--for example, early premalignant lesions such as cervical intraepithelial neoplasia, the fact that some tumors very rarely become malignant, the observation of plateaux in the growth of some solid tumors, and, finally, long lag phases of growth until mutations arise that eventually result in exponential growth.

Neurotrophin 3 rescues neuronal precursors from apoptosis and promotes neuronal differentiation in the embryonic metanephric kidney.

We analyzed the developmental regulation and role of the neurotrophins during metanephric kidney morphogenesis. RNase protection assay revealed the presence of nerve growth factor, neurotrophin 3 (NT-3), and brain-derived neurotrophic factor mRNAs and the regulation of their expression during embryonic development of rat metanephros. NT-3 induced differentiation (neurite outgrowth) and survival (inhibition of apoptosis) of the neuronal precursors in cultured nephrogenic mesenchymes and neuronal differentiation in cultured whole kidneys, whereas NT-4/5, brain-derived neurotrophic factor, and nerve growth factor were without effect. The neurotrophins did not trigger tubular differentiation of isolated nephrogenic cells, which underwent apoptosis when cultured with or without the neurotrophins. NT-3 is thus an inducer of differentiation and a survival factor for renal neuronal cells, but none of the neurotrophins is a morphogen in kidney tubule induction.

Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.

Barley telomeres shorten during differentiation but grow in callus culture.

Eukaryotic chromosomes terminate with long stretches of short, guanine-rich repeats. These repeats are added de novo by a specialized enzyme, telomerase. In humans telomeres shorten during differentiation, presumably due to the absence of telomerase activity in somatic cells. This phenomenon forms the basis for several models of telomere role in cellular senescence. Barley (Hordeum vulgare L.) telomeres consist of thousands of TTTAGGG repeats, closely resembling other higher eukaryotes. In vivo differentiation and aging resulted in reduction of terminal restriction fragment length paralleled by a decrease of telomere repeat number. Dedifferentiation in callus culture resulted in an increase of the terminal restriction fragment length and in the number of telomere repeats. Long-term callus cultures had very long telomeres. Absolute telomere lengths were genotype dependent, but the relative changes due to differentiation, dedifferentiation, and long-term callus culture were consistent among genotypes. A model is presented to describe the potential role of the telomere length in regulation of a cell's mitotic activity and senescence.

Autocrine/paracrine role of insulin-related growth factors in neurogenesis: local expression and effects on cell proliferation and differentiation in retina.

Early neurogenesis progresses by an initial massive proliferation of neuroepithelial cells followed by a sequential differentiation of the various mature neural cell types. The regulation of these processes by growth factors is poorly understood. We intend to understand, in a well-defined biological system, the embryonic chicken retina, the role of the insulin-related growth factors in neurogenesis. We demonstrate the local presence of signaling elements together with a biological response to the factors. Neuroretina at days 6-8 of embryonic development (E6-E8) expressed proinsulin/insulin and insulin-like growth factor I (IGF-I) mRNAs as well as insulin receptor and IGF type I receptor mRNAs. In parallel with this in vivo gene expression, E5 cultured neuroretinas synthesized and released to the medium a metabolically radiolabeled immunoprecipitable insulin-related peptide. Furthermore, insulin-related immunoreactive material with a HPLC mobility close to that of proinsulin was found in the E6-E8 vitreous humor. Exogenous chicken IGF-I, human insulin, and human proinsulin added to E6 cultured neuroretinas showed relatively close potencies stimulating proliferation, as determined by [methyl-3H]thymidine incorporation, with a plateau reached at 10(-8) M. These factors also stimulated neuronal differentiation, indicated by the expression of the neuron-specific antigen G4. Thus, insulin-related growth factors, interestingly including proinsulin, are present in the developing chicken retina and appear to play an autocrine/paracrine stimulatory role in the progression of neurogenesis.

Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

Glial cell line-derived neurotrophic factor promotes the survival and morphologic differentiation of Purkinje cells.

Glial cell line-derived neurotrophic factor (GDNF) promotes survival of midbrain dopaminergic neurons and motoneurons. Expression of GDNF mRNA in cerebellum raises the possibility that cells within this structure might also respond to GDNF. To examine potential trophic activities of GDNF, dissociated cultures of gestational day 18 rat cerebellum were grown for < or = 21 days in the presence of factor. GDNF increased Purkinje cell number without affecting the overall number of neurons or glial cells. A maximal response (50% above control) was elicited with GDNF at 1 pg/ml. Effects of GDNF on Purkinje cell differentiation were examined by scoring the morphologic maturation of cells in treated and control cultures. GDNF increased the proportion of Purkinje cells that displayed relatively mature morphologies, characterized by dendritic thickening and the development of spines and filopodial extensions. Morphologic maturation of the overall neuronal population was unaffected. In sum, our data indicate that GDNF is a potent survival and differentiation factor for Purkinje cells, the efferent neurons of cerebellar cortex. Together with its other actions, these findings raise the possibility that GDNF might be a critical trophic factor at multiple loci in neuronal circuits that control motor function.

Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation.

Treatment of the human promyelocytic leukemia cell line HL-60 with antisense oligodeoxynucleotides to UDP-N-acetylgalactosamine:beta-1,4-N-acetylgalactosaminyl-transferase (GM2-synthase; EC 2.4.1.92) and CMP-sialic acid:alpha-2,8-sialyltransferase (GD3-synthase; EC 2.4.99.8) sequences effectively down-regulated the synthesis of more complex gangliosides in the ganglioside synthetic pathways after GM3, resulting in a remarkable increase in endogenous GM3 with concomitant decreases in more complex gangliosides. The treated cells underwent monocytic differentiation as judged by morphological changes, adherent ability, and nitroblue tetrazolium staining. These data provide evidence that the increased endogenous ganglioside GM3 may play an important role in regulating cellular differentiation and that the antisense DNA technique proves to be a powerful tool in manipulating glycolipid synthesis in the cell.

A 20-nucleotide element in the intestinal fatty acid binding protein gene modulates its cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of expression in transgenic mice.

A sequence of epithelial cell proliferation, allocation to four principal lineages, migration-associated differentiation, and cell loss occurs along the crypt-villus axis of the mouse intestine. The sequence is completed in a few days and is recapitulated throughout the life-span of the animal. We have used an intestine-specific fatty acid binding protein gene, Fabpi, as a model for studying regulation of gene expression in this unique developmental system. Promoter mapping studies in transgenic mice identified a 20-bp cis-acting element (5'-AGGTGGAAGCCATCACACTT-3') that binds small intestinal nuclear proteins and participates in the control of Fabpi's cephalocaudal, differentiation-dependent, and cell lineage-specific patterns of expression. Immunocytochemical studies using confocal and electron microscopy indicate that it does so by acting as a suppressor of gene expression in the distal small intestine/colon, as a suppressor of gene activation in proliferating and nonproliferating cells located in the crypts of Lieberkühn, and as a suppressor of expression in the growth factor and defensin-producing Paneth cell lineage. The 20-bp domain has no obvious sequence similarities to known transcription factor binding sites. The three functions modulated by this compact element represent the types of functions required to establish and maintain the intestine's remarkably complex spatial patterns of gene expression. The transgenes described in this report also appear to be useful in characterizing the crypt's stem cell hierarchy.

Development in one dimension: the rapid differentiation of Dictyostelium discoideum in glass capillaries.

When Dictyostelium discoideum cells are drawn into a fine glass capillary, they rapidly begin the first steps toward the formation of prestalk and prespore zones. Some of the events occur within a minute or two, whereas others follow later. The cells in the front segment are actively motile and those in the hind segment are passive. The volumes of the segments are proportional for different-sized cell masses, and those proportions are the same as those found in normal slugs. When the cells are stained with the vital dye neutral red, the anterior zone becomes darker simultaneously with the formation of the division line. Green fluorescent protein expressed from a stalk-specific promoter is synthesized mostly in the anterior end. Later, this capillary prestalk zone shows a sharp increase in alkaline phosphatase activity, which is known to be characteristic of prestalk cells.

Contrasting roles for Myc and Mad proteins in cellular growth and differentiation.

The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state.

Overexpression of DR-nm23, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells.

Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.