Morphogenetic Effects of Neuregulin (Neu Differentiation Factor) in Cultured Epithelial Cells

Neuregulin, or neu differentiation factor, induces cell proliferation or differentiation through interaction with members of the ErbB family of receptor tyrosine kinases. We report that neuregulin can also induce profound morphogenic responses in cultured epithelial cells of different origins. These effects include scattering of small epithelial islands and rearrangement of larger cell islands into ordered ring-shaped arrays with internal lumens. The ring-forming cells are interconnected by cadherin- and β-catenin-containing adherens junctions. In confluent cultures, neuregulin treatment induces formation of circular lumenlike gaps in the monolayer. Both cell scattering and ring formation are accompanied by a marked increase in cell motility that is independent of hepatocyte growth factor/scatter factor and its receptor (c-Met). Affinity-labeling experiments implied that a combination of ErbB-2 with ErbB-3 mediates the morphogenic signal of neuregulin in gastric cells. Indeed, a similar morphogenic effect could be reconstituted in nonresponsive cells by coexpression of ErbB-2 and -3. We conclude that a heterodimer between the kinase-defective neuregulin receptor, ErbB-3, and the coreceptor, ErbB-2, mediates the morphogenetic action of neuregulin.

Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 receptor pathway

Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the transforming growth factor β superfamily, progesterone receptor, cyclooxygenase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E2 (PGE2) receptor (EP2; Ptgerep2) are required for fertility in female but not male mice. To define the interrelationship of these factors, we used a preovulatory granulosa cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimulated Cox2 mRNA within 2 h, and PGE2 within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of GDF-9 but that progesterone synthesis required an intermediate. To determine whether prostaglandin synthesis was required for progesterone production, we analyzed the effects of PGE2 and cyclooxygenase inhibitors on this process. PGE2 can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fold increase over 24 h, respectively). Furthermore, indomethacin or NS-398, inhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE2 to cultures containing both GDF-9 and NS-398 overrides the NS-398 block in progesterone synthesis. To further define the PGE2-dependent pathway, we show that butaprost, a specific EP2 agonist, stimulates progesterone synthesis and overrides the NS-398 block. In addition, GDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone-independent pathway. Thus, GDF-9 induces an EP2 signal transduction pathway which appears to be required for progesterone synthesis in cumulus granulosa cells. These studies further demonstrate the importance of oocyte–somatic cell interactions in female reproduction.

Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene

Tumor necrosis factor-related, activation-induced cytokine (TRANCE), a tumor necrosis factor family member, mediates survival of dendritic cells in the immune system and is required for osteoclast differentiation and activation in the skeleton. We report the skeletal phenotype of TRANCE-deficient mice and its rescue by the TRANCE transgene specifically expressed in lymphocytes. TRANCE-deficient mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation at several skeletal sites, including the limbs, skull, and vertebrae. These mice had marked chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes. Transgenic overexpression of TRANCE in lymphocytes of TRANCE-deficient mice rescued osteoclast development in two locations in growing long bones: excavation of marrow cavities permitting hematopoiesis in the marrow spaces, and remodeling of osteopetrotic woven bone in the shafts of long bones into histologically normal lamellar bone. However, osteoclasts in these mice failed to appear at the chondroosseous junction and the metaphyseal periosteum of long bones, nor were they present in tooth eruption pathways. These defects resulted in sclerotic metaphyses with persistence of club-shaped long bones and unerupted teeth, and the growth plate defects were largely unimproved by the TRANCE transgene. Thus, TRANCE-mediated regulation of the skeleton is complex, and impacts chondrocyte differentiation and osteoclast formation in a manner that likely requires local delivery of TRANCE.

Leukemia inhibitory factor induces differentiation of pituitary corticotroph function: an immuno-neuroendocrine phenotypic switch.

Leukemia inhibitory factor (LIF) promotes differentiated cell function in several systems. We recently reported LIF and LIF receptor expression in human fetal pituitary corticotrophs in vivo and demonstrated LIF stimulation of adrenocorticotrophin (ACTH) transcription in vitro, suggesting a role for LIF in corticotroph development. We therefore assessed the action of LIF on proliferating murine corticotroph cells (AtT20). LIF impairs proliferation of AtT20 cells (25% reduction versus control, P < 0.03), while simultaneously enhancing ACTH secretion (2-fold, P < 0.001) and augmenting ACTH responsiveness to corticotrophin-releasing hormone (CRH) action (4-fold, P < 0.001). This attenuation of cell growth is due to a block of cell cycle progression from G1 into S phase, as measured by flow cytometric analysis (24 +/- 0.8 versus 11.57 +/- 1.5, P < 0.001). Using bromodeoxyuridine incorporation assays, loss of cells in S phase was confirmed (25 +/- 0.08 to 9.4 +/- 1.4, P < 0.008). In contrast, CRH induced the G2/M phase (3.6 +/- 0.2 to 15.4 +/- 3, P < 0.001). This effect was blunted by LIF (P < 0.001 versus CRH alone). Cyclin A mRNA levels, which decline in S phase, were stimulated 3.5-fold by LIF and markedly suppressed by CRH. These results indicate a LIF-induced cell cycle block occurring at G1/S in corticotroph cells. Thus, LIF reduces proliferation, enhances ACTH secretion, and potentiates effects of CRH on ACTH secretion while blocking effects of CRH on the cell cycle. Responses of these three markers of differentiated corticotroph function indicate LIF to be a differentiation factor for pituitary corticotroph cells by preferential phenotypic switching from proliferative to synthetic.

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.

Opposing roles of pRB and p107 in adipocyte differentiation

The retinoblastoma (RB) family of proteins, pRB, p107, and p130, have been postulated to be partially redundant in their ability to regulate progression through the G1 phase of the cell cycle. However, pRB appears to be unique in its capacity as a classical tumor suppressor, possibly because of a specialized role in maintaining the balance between proliferation and differentiation. A variety of studies have in fact revealed an apparent role for pRB in cellular differentiation and development. However, roles for p107 and p130 in differentiation have not yet been established, and knockout mouse studies have indicated that they may be functionally redundant during development, and possibly perform a role in differentiation distinct from that of pRB. Using adipogenesis as a model, we have indeed found distinct roles for the pRB family proteins in regulating differentiation. 3T3 fibroblasts deficient in p107 and p130 differentiate with high efficiency, whereas pRB−/− 3T3 cells exhibit defects in their differentiation potential. Moreover, over-expression of pRB in wild-type cells promotes differentiation, whereas over-expression of p107 antagonizes differentiation. The seemingly opposing roles of pRB family members in adipocyte differentiation can be explained, at least in part, by a requirement for pRB in maintaining cell cycle exit as well as potentiating the activity of the differentiation-associated transcription factor, C/EBPα. p107 does not affect C/EBPα-driven transcription and is not required for cell cycle exit, but instead, loss of p107 lowers the requirement for the differentiation factor PPARγ. These findings suggest contrasting biological roles for individual members of the pRB family of proteins that may explain why pRB, but not p107, is commonly mutated during human tumor development.

ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response

The ATM gene is mutated in the syndrome of ataxia telangiectasia (AT), associated with neurologic dysfunction, growth abnormalities, and extreme radiosensitivity. Insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and inhibition of apoptosis. We report here that AT cells express low levels of IGF-IR and show decreased IGF-IR promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and elevated IGF-IR levels, whereas expression in wild-type cells of a dominant negative fragment of ATM specifically reduces IGF-IR expression, results consistent with a role for ATM in regulating IGF-IR expression at the level of transcription. When expression of IGF-IR cDNA is forced in AT cells via a heterologous viral promoter, near normal radioresistance is conferred on the cells. Conversely, in ATM cells complemented with the ATM cDNA, specific inhibition of the IGF-IR pathway prevents correction of the radiosensitivity. Taken together, these results establish a fundamental link between ATM function and IGF-IR expression and suggest that reduced expression of IGF-IR contributes to the radiosensitivity of AT cells. In addition, because IGF-I plays a major role in human growth and metabolism and serves as a survival and differentiation factor for developing neuronal tissue, these results may provide a basis for understanding other aspects of the AT syndrome, including the growth abnormalities, insulin resistance, and neurodegeneration.

IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor γ1

IL-4 is a pleiotropic immune cytokine secreted by activated TH2 cells that inhibits bone resorption both in vitro and in vivo. The cellular targets of IL-4 action as well as its intracellular mechanism of action remain to be determined. We show here that IL-4 inhibits receptor activator of NF-κB ligand-induced osteoclast differentiation through an action on osteoclast precursors that is independent of stromal cells. Interestingly, this inhibitory effect can be mimicked by both natural as well as synthetic peroxisome proliferator-activated receptor γ1 (PPARγ1) ligands and can be blocked by the irreversible PPARγ antagonist GW 9662. These findings suggest that the actions of IL-4 on osteoclast differentiation are mediated by PPARγ1, an interpretation strengthened by the observation that IL-4 can activate a PPARγ1-sensitive luciferase reporter gene in RAW264.7 cells. We also show that inhibitors of enzymes such as 12/15-lipoxygenase and the cyclooxygenases that produce known PPARγ1 ligands do not abrogate the IL-4 effect. These findings, together with the observation that bone marrow cells from 12/15-lipoxygenase-deficient mice retain sensitivity to IL-4, suggest that the cytokine may induce novel PPARγ1 ligands. Our results reveal that PPARγ1 plays an important role in the suppression of osteoclast formation by IL-4 and may explain the beneficial effects of the thiazolidinedione class of PPARγ1 ligands on bone loss in diabetic patients.

Dissociation between bone resorption and bone formation in osteopenic transgenic mice

Bone mass is maintained constant in vertebrates through bone remodeling (BR). BR is characterized by osteoclastic resorption of preexisting bone followed by de novo bone formation by osteoblasts. This sequence of events and the fact that bone mass remains constant in physiological situation lead to the assumption that resorption and formation are regulated by each other during BR. Recent evidence shows that cells of the osteoblastic lineage are involved in osteoclast differentiation. However, the existence of a functional link between the two activities, formation and resorption, has never been shown in vivo. To define the role of bone formation in the control of bone resorption, we generated an inducible osteoblast ablation mouse model. These mice developed a reversible osteopenia. Functional analyses showed that in the absence of bone formation, bone resorption continued to occur normally, leading to an osteoporosis of controllable severity, whose appearance could be prevented by an antiresorptive agent. This study establishes that bone formation and/or bone mass do not control the extent of bone resorption in vivo.

β-Neuregulin-1 is required for the in vivo development of functional Ca2+-activated K+ channels in parasympathetic neurons

The development of functional Ca2+-activated K+ channels (KCa) in chick ciliary ganglion (CG) neurons requires interactions with afferent preganglionic nerve terminals. Here we show that the essential preganglionic differentiation factor is an isoform of β-neuregulin-1. β-Neuregulin-1 transcripts are expressed in the midbrain preganglionic Edinger–Westphal nucleus at developmental stages that coincide with or precede the normal onset of macroscopic KCa in CG neurons. Injection of β-neuregulin-1 peptide into the brains of developing embryos evoked a robust stimulation of functional KCa channels at stages before the normal appearance of these channels in CG neurons developing in vivo. Conversely, injection of a neutralizing antiserum specific for β-neuregulin-1 inhibited the development of KCa channels in CG neurons. Low concentrations of β-neuregulin-1 evoked a robust increase in whole-cell KCa in CG neurons cocultured with iris target tissues. By contrast, culturing CG neurons with iris cells or low concentrations of β-neuregulin-1 by themselves was insufficient to stimulate KCa. These data suggest that the preganglionic factor required for the development of KCa in ciliary ganglion neurons is an isoform of β-neuregulin-1, and that this factor acts in concert with target-derived trophic molecules to regulate the differentiation of excitability.

The retinoblastoma protein binds to RIZ, a zinc-finger protein that shares an epitope with the adenovirus E1A protein.

The retinoblastoma protein (Rb) is a target of viral oncoproteins. To explore the hypothesis that viral proteins may be structural mimics of cellular proteins, we have searched cDNA libraries for Rb-binding proteins. We report here the cloning of a cDNA for the protein RIZ from rat and human cells. RIZ is a 250-kDa nuclear protein containing eight zinc-finger motifs. It contains an Rb-binding motif that shares an antigenic epitope with the C terminus of E1A. A domain is conserved between RIZ and the PRDI-BF1/Blimp-1 differentiation factor. Other motifs of RIZ include putative GTPase and SH3 (src homology domain 3) domains. RIZ is preferentially expressed in both adult and embryonic rat neuroendocrine tissues. It is also expressed in human retinoblastoma cells and at low levels in all other human cell lines examined. While the function of RIZ is not yet clear, its structure and pattern of expression suggest a role for RIZ in transcriptional regulation during neuronal differentiation and pathogenesis of retinoblastoma.

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.

Conditional expression of the ubiquitous transcription factor MafK induces erythroleukemia cell differentiation.

Transcription factor NF-E2 activity is thought to be crucial for the transcriptional regulation of many erythroid-specific genes. The three small Maf family proteins (MafF, MafG, and MafK) that are closely related to the c-Maf protooncoprotein constitute half of the NF-E2 activity by forming heterodimers with the large tissue-restricted subunit of NF-E2 called p45. We have established and characterized murine erythroleukemia cells that conditionally overexpress MafK from a metallothionein promoter. The conditional expression of MafK caused accumulation of hemoglobin, an indication of terminal differentiation along the erythroid pathway. Concomitantly, DNA binding activities containing MafK were induced within the MafK-overexpressing cells. These results demonstrate that MafK can promote the erythroid differentiation program in erythroleukemia cells and suggest that the small Maf family proteins are key regulatory molecules for erythroid differentiation.

Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development

The vertebrate lens is a tissue composed of terminally differentiated fiber cells and anterior lens epithelial cells. The abundant, preferential expression of the soluble proteins called crystallins creates a transparent, refractive index gradient in the lens. Several transcription factors such as Pax6, Sox1, and L-Maf have been shown to regulate lens development. Here we show that mice lacking the transcription factor c-Maf are microphthalmic secondary to defective lens formation, specifically from the failure of posterior lens fiber elongation. The marked impairment of crystallin gene expression observed is likely explained by the ability of c-Maf to transactivate the crystallin gene promoter. Thus, c-Maf is required for the differentiation of the vertebrate lens.