Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.

To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, we have targeted the mouse Cdk4 locus by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types.

Hematopoietic cytokine receptor signaling.

Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Proper culmination of these diverse pathways forms the basis for an orderly generation of different cell types. Recent studies conducted over the past 10-15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors termed STATs (signal transducers and activators of transcription). Aberration in these pathways, such as that caused by the recently identified JAK2V617F mutation, is an underlying cause for diseases such as leukemias and other myeloproliferative disorders. This recent discovery, when coupled with the fact that STATs are activated by oncoproteins such as BCR-ABL, underscores the importance of the JAK-STAT pathway in both normal cellular development and disease states.

Opioid peptide modulation of Ca(2+)-dependent K+ and voltage-activated Ca2+ currents in bovine adrenal chromaffin cells.

Opioid peptides are abundantly expressed in the adrenal medulla, and there is evidence that they may be released presynaptically or as medullary paracrine agents. To assess the physiological relevance of these observations, we investigated opioid effects on ionic currents from cultured bovine adrenal chromaffin cells. Under whole-cell path-clamp conditions, opioid peptides, acting via a mu-type opioid receptor, strongly potentiated the large conductance Ca(2+)-dependent K+ (BK) channel current. Opioids also inhibited voltage-activated Ca2+ currents. Application of opioid peptides to the extracellular face of outside-out patches also increased opening activity of single BK channels, suggestive of tight receptor-channel coupling. This potentiating effect on BK current, combined with the inhibition of Ca2+ current, indicates that opioids may have an inhibitory influence on secretory activity of the adrenal medulla. The widespread distribution of the BK channel class suggests that the significance of its modulation by opioids could also extend beyond the adrenal gland.

Specific inhibitors of protein kinase C block transmitter-induced modulation of sensory neuron calcium current.

Modulation of neuronal, voltage-dependent calcium current has been described for a number of transmitters and peptides, but the biochemical basis for this phenomenon has not been completely identified. In several cases protein kinase C (PKC) is thought to mediate transmitter inhibition of calcium current; however, a lack of specific PKC inhibitors has hampered a direct physiological test of this idea. We have used the whole-cell, tight-seal configuration of the patch-clamp technique to apply intracellularly two specific PKC inhibitors to the cell bodies of embryonic chick sensory neurons. Both inhibitors, a 17 kd protein purified from bovine brain and a synthetic 13 amino acid "pseudosubstrate" peptide, blocked inhibition of calcium current by either norepinephrine or an exogenously applied PKC activator. These results provide strong evidence that activation of PKC is a prerequisite for the modulation of sensory neuron calcium current by norepinephrine.

Granulocyte colony-stimulating factor-induced upregulation of Jak3 transcription during granulocytic differentiation is mediated by the cooperative action of Sp1 and Stat3.

We previously demonstrated that Jak3 is a primary response gene for G-CSF and ectopic overexpression of Jak3 can accelerate granulocytic differentiation of normal mouse bone marrow cells induced by G-CSF and GM-CSF. To gain insight into the regulation of G-CSF-induced transcription of Jak3, we constructed deletion and linker scanning mutants of the Jak3 promoter sequences and performed luciferase reporter assays in the murine myeloid cell line 32Dcl3, with and without G-CSF stimulation. These experiments showed that mutation of a -67 to -85 element, which contained a putative Sp1 binding site, or mutation of a -44 to -53 GAS element resulted in a marked reduction of Jak3 promoter activity. Electrophoretic mobility shift assays revealed that Sp1 and Stat3 present in nuclear lysates of 32Dcl3 cells stimulated with G-CSF can bind to the -67 to -85 element and -44 to -53 GAS element, respectively. In addition, cotransfection of a constitutively active mutant of Stat3 along with a Jak3 promoter/luciferase reporter resulted in enhanced Jak3 promoter activity. Together, these results demonstrate that activation of Jak3 transcription during G-CSF- induced granulocytic differentiation is mediated by the combined action of Sp1 and Stat3, a mechanism also shown to be important in IL-6-induced monocytic differentiation.

Structure and function of a virally encoded fungal toxin from Ustilago maydis: a fungal and mammalian Ca2+ channel inhibitor.

BACKGROUND: The P4 strain of the corn smut fungus, Ustilago maydis, secretes a fungal toxin, KP4, encoded by a fungal virus (UMV4) that persistently infects its cells. UMV4, unlike most other (non-fungal) viruses, does not spread to uninfected cells by release into the extracellular milieu during its normal life cycle and is thus dependent upon host survival for replication. In symbiosis with the host fungus, UMV4 encodes KP4 to kill other competitive strains of U. maydis, thereby promoting both host and virus survival. KP4 belongs to a family of fungal toxins and determining its structure should lead to a better understanding of the function and evolutionary origins of these toxins. Elucidation of the mechanism of toxin action could lead to new anti-fungal agents against human pathogens. RESULTS: We have determined the atomic structure of KP4 to 1.9 A resolution. KP4 belongs to the alpha/beta-sandwich family, and has a unique topology comprising a five-stranded antiparallel beta-sheet with two antiparallel alpha-helices lying at approximately 45 degrees to these strands. The structure has two left-handed beta alpha beta cross-overs and a basic protuberance extending from the beta-sheet. In vivo experiments demonstrated abrogation of toxin killing by Ca2+ and, to a lesser extent, Mg2+. These results led to experiments demonstrating that the toxin specifically inhibits voltage-gated Ca2+ channels in mammalian cells. CONCLUSIONS: Similarities, although somewhat limited, between KP4 and scorpion toxins led us to investigate the possibility that the toxic effects of KP4 may be mediated by inhibition of cation channels. Our results suggest that certain properties of fungal Ca2+ channels are homologous to those in mammalian cells. KP4 may, therefore, be a new tool for studying mammalian Ca2+ channels and current mammalian Ca2+ channel inhibitors may be useful lead compounds for new anti-fungal agents.

JAK3: a novel JAK kinase associated with terminal differentiation of hematopoietic cells.

The Janus Kinases (JAK) JAK1, JAK2, and TYK2 are protein tyrosine kinases which play a pivotal role in the signal transduction process mediated by cytokines. These kinases appear to transduce signals via their substrates which modulate programs of gene expression specific to the respective signals. It is becoming increasingly evident that certain cytokines such as Granulocyte Colony Stimulating Factor (GCSF) can transmit signals for both cellular proliferation and differentiation. It is at present unclear whether both of these signals are transmitted by the same JAK kinase or whether an entire family of such kinases are involved in this process. To determine if additional members of JAK kinase family exist, we designed a polymerase chain reaction based strategy which resulted in the identification of a new member of the JAK kinase family. This new kinase, which we have named JAK3 is encoded by a 4.3 kb mRNA transcript. Nucleotide sequence analysis of a full length cDNA derived from this mRNA revealed that it encodes an open reading frame of 3897 bp. The protein encoded by this mRNA contains the double catalytic domain characteristic of the JAK family kinases. The most striking difference between JAK3 and the other JAK kinases is the presence of two stretches of additional amino acid sequence of 147 and 28 residues which span between amino acid positions 322 to 469 and 632 to 660 respectively. Expression studies indicate that JAK3 is expressed at very low levels in immature hematopoietic cells, but its expression is dramatically up-regulated during terminal differentiation of these cells. These results suggest that JAK3 plays an important role in the differentiation of hematopoietic cells.

Janus kinases: components of multiple signaling pathways.

Cytoplasmic Janus protein tyrosine kinases (JAKs) are crucial components of diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Evidence to date, indicates that JAK kinase function may integrate components of diverse signaling cascades. While it is likely that activation of STAT proteins may be an important function attributed to the JAK kinases, it is certainly not the only function performed by this key family of cytoplasmic tyrosine kinases. Emerging evidence indicates that phosphorylation of cytokine and growth factor receptors may be the primary functional attribute of JAK kinases. The JAK-triggered receptor phosphorylation can potentially be a rate-limiting event for a successful culmination of downstream signaling events. In support of this hypothesis, it has been found that JAK kinase function is required for optimal activation of the Src-kinase cascade, the Ras-MAP kinase pathway, the PI3K-AKT pathway and STAT signaling following the interaction of cytokine/interferon receptors with their ligands. Aberrations in JAK kinase activity, that may lead to derailment of one or more of the above mentioned pathways could disrupt normal cellular responses and result in disease states. Thus, over-activation of JAK kinases has been implicated in tumorigenesis. In contrast, loss of JAK kinase function has been found to result in disease states such as severe-combined immunodeficiency. In summary, optimal JAK kinase activity is a critical determinant of normal transmission of cytokine and growth factor signals.

Cell cycle control of pancreatic beta cell proliferation.

Diabetes mellitus ensues as a consequence of the body's inability to respond normally to high blood glucose levels. The onset of diabetes is due to several pathological changes, which are a reflection of either the inability of the pancreatic beta cells to secrete sufficient insulin to combat the hyperglycemia or a state of insulin resistance in target tissues. However, the significance of changes in beta cell mass and decreased beta cell proliferation or growth in progression of diabetes has been under-appreciated. Beta cells, like all other cells of our body are under the regulatory checks and balances enforced by changes in cell cycle progression. However, very little is known regarding the key components of the cell cycle machinery regulating cell cycle control of beta cells. Knowledge of key elements involved in cell cycle regulation of beta cells will go a long way in improving our understanding of the replication capacity and developmental biology of beta cells. This information is essential for us to design new approaches that can be used to correct beta cell deficiency in diabetes. This review focuses on the current knowledge of factors important for proliferation of beta cells and proposes a cell cycle model for regeneration of the beta cell population lost or reduced in diabetes.

Smad3 deficiency inhibits v-ras-induced transformation by suppression of JNK MAPK signaling and increased farnesyl transferase inhibition.

The ability of transforming growth factor-beta (TGF-beta) to modulate various effects on distinct cell lineages has been a central feature of its multi-faceted nature. The purpose of this study was to access the effects of deletion of a key TGF-beta signal transducer, Smad3, on MAPK activation and v-Ras(Ha)-transformation of primary mouse embryonic fibroblasts (MEFs). We observe reduced TGF-beta1 and v-ras(Ha) mediated activation of the JNK and ERK MAPK pathway upon ablation of Smad3. Further, Smad3-deficient MEFs demonstrate resistance to v-ras(Ha)-induced transformation while the absence of Smad3 results in increased inhibition of farnesyl transferase activity. Taken together, these observations demonstrate that the absence of Smad3 protects fibroblasts from oncogenic transformation by (i) augmenting farnesyl transferase inhibition and (ii) suppressing the Ras-JNK MAPK pathway. These results provide new insights into the molecular mechanisms involved in v-Ras(Ha) oncogene-induced mesenchymal phenotypic transformation.

Aberrant nucleocytoplasmic localization of the retinoblastoma tumor suppressor protein in human cancer correlates with moderate/poor tumor differentiation.

Inactivation of the retinoblastoma (RB) tumor suppressor pathway, via elevated cyclin-dependent kinase (CDK) activity, is observed in majority of human cancers. Since CDK deregulation is evident in most cancer cells, pharmacological CDK inhibition has become an attractive therapeutic strategy in oncology. We recently showed that an oncogenic CDK4(R24C) mutation alters the subcellular localization of the normally nuclear RB phosphoprotein. Here, using 71 human cancer cell lines and over 300 primary human cancer tissues, we investigated whether changes in RB subcellular localization occur during human cancer progression. We uncover that diverse human cancers and their derived cell lines, particularly those with poor tumor differentiation, display significant cytoplasmic mislocalization of ordinarily nuclear RB. The nucleocytoplasmically distributed RB was derived via CDK-dependent and Exportin1-mediated nuclear export. Indeed, cytoplasmically mislocalized RB could be efficiently confined to the nucleus by pharmacologically reducing CDK activity or by inhibiting the Exportin1-mediated nuclear export pathway. Our observations uncover a post-translational CDK-dependent mechanism of RB inactivation and suggest that cytoplasmically localized RB may harbor a tumor promoting function. We propose that RB inactivation, via aberrant nucleocytoplasmic transport, may disrupt normal cell differentiation programs and accelerate the cancer process. These results are evidence that tumor cells modulate the protein transport machinery thereby making the protein transport process a viable therapeutic target.

A Ca2(+)-activated K+ current in ras-transformed fibroblasts is absent from nontransformed cells.

Biochemical similarities between ras proteins and the GTP-binding proteins and correlation of ras-induced cell transformation with altered transmembrane cation fluxes indicate that ras proteins may act to modulate ion channel activity. To test this idea, whole cell, tight-seal, patch-clamp recording was used to compare macroscopic currents of ras-transformed fibroblasts with currents of their nontransformed counterparts. A prominent calcium-activated, voltage-independent potassium current was observed in 83-100% of cells from three separate fibroblast lines transformed by two different oncogenic ras alleles, whereas the same current was present at much smaller amplitudes in only 0-15% of nontransformed cells. The calcium-activated potassium current is blocked by charybdotoxin and by concentrations of tetraethylammonium above 1 mM, but it is insensitive to apamin. Both normal and ras-transformed cells have another calcium-activated current that is not potassium selective, and, consistent with other studies, normal cells display a voltage-activated calcium conductance. These results suggest that the mechanisms by which ras triggers or maintains cell transformation may involve alterations in the number or activity of certain ion channels, in particular, a type of calcium-activated potassium channel.

Neuromuscular synaptic transmission in Limulus polyphemus--II. Release of amino acid putative transmitters from the neuromuscular preparation.

High-performance liquid chromatography with fluorescence detection was used to assay the release of putative amino acid transmitters from the Limulus neuromuscular preparation. Motor axon stimulation increased the concentrations of aspartate, glutamate and eight other amino acids in fluid bathing the neuromuscular preparation. Pentobarbital, which attenuates the excitatory postsynaptic potential of Limulus muscle, was used to block both synaptic activation of muscle fibers and any amino acid release that may have resulted from this activation. Stimulus-induced release of glutamate and five other amino acids was blocked by pentobarbital, while release of aspartate and three other amino acids was unaffected; a result which suggests that the latter group of amino acids was released presynaptically. Aspartate is the only physiologically active compound in this group. Consideration is given both to the difficulties involved in interpreting sites of amino acid release and to the problem of using pentobarbital as a presumed postsynaptic antagonist. The evidence concerning the relative merits of either aspartate or glutamate as the natural excitatory transmitter at the Limulus neuromuscular junction is discussed.

The growth regulatory fibroblast IK channel is the prominent electrophysiological feature of rat prostatic cancer cells.

Physiological effectors for mitogenic cell growth control remain to be determined for mammalian tumor cells, particularly those derived from prostatic tissue. One such effector for mitogenic Ras/MAPK signaling in fibroblasts is an intermediate-conductance, calcium-activated potassium channel (FIK). In this study patch-clamp electrophysiology was used to show that both AT2.1 and MatLyLu rat prostate cancer cell lines express high levels of a current identified as FIK, based on the following criteria: activation by elevation of intracellular calcium, voltage independence, potassium selectivity, and block by charybdotoxin (ChTX) and the Stichodactyla helianthus potassium channel neurotoxin (StK). FIK current densities in AT2.1 and MatLyLu cells were comparable to the high levels seen in fibroblasts transfected with oncogenic Ras or Raf, suggesting hyperactivity of the Ras/MAPK pathway in prostatic cancer cells. Voltage-gated sodium current was present in most MatLyLu cells but absent from AT2.1 cells, and all AT2.1 cells had voltage-gated potassium currents. Thus, FIK is the main electrophysiological feature of rat prostatic cancer cells as it is for mitogenically active fibroblasts, suggesting it may play a similar growth regulatory role in both.

Nucleotide-independent modulation of Ca(2+)-dependent K+ channel current by a mu-type opioid receptor.

Physiological responses to opiates and opioid peptides are transduced via receptors coupled to G proteins. The effectors for these G proteins are often ion channels or second messenger systems that modulate channel activity. In cultured bovine adrenal medullary chromaffin cells (BAMCCs), the activity of a calcium-dependent, voltage-sensitive, potassium (BK) channel is robustly potentiated by a mu-type opioid receptor, an effect consistent with the inhibitory role of opioids versus neural excitability. Patch-clamp electrophysiology was used to investigate coupling between the mu receptor and BK channel, leading to rather surprising results. Potentiation of BK channel activity by the mu-selective agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (10 nM) was unaffected by all attempts to disrupt or alter G protein function, including incubation of cells with pertussis toxin (PTX) and inclusion of guanosine 5'-O-(2-thio)diphosphate (GDP beta S) or guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) in intracellular recording solutions. However, dopamine D2 receptor potentiation of BK current in these same cells was affected by PTX, GDP beta S, and GTP gamma S in predictable fashion. Thus, PTX and GDP beta S inhibited dopamine potentiation of BK current, and GTP gamma S prolonged reversal of dopamine action. These results suggest that the BAMCC BK channel is not coupled to the mu receptor via a GTP-dependent mechanism, whereas in the same cells the dopamine D2 receptor modulates BK channel activity in a conventional GTP-dependent manner. In addition, replacement of both ATP and GTP with nonhydrolyzable analogs also failed to affect either potentiation or recovery of BK channel activity in response to [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin. These results indicate that in BAMCCs the mu-opioid receptor modulates BK channel activity independently of either G proteins or phosphorylation-dependent processes.

Fibroblast growth factor-induced increases in calcium currents in the PC12 pheochromocytoma cell line are tyrosine phosphorylation dependent.

The PC12 rat pheochromocytoma cell line is widely used to study neuronal differentiation by growth factors. In response to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), PC12 cells differentiate into sympathetic-like neurons and become electrically excitable. Using whole cell patch-clamp recording, with barium as a charge carrier, we looked at the effects of bFGF on calcium channel expression as reflected by changes in barium current amplitudes normalized to cell membrane area. Similar to the effect reported for NGF, we show that 7 day treatment with bFGF increased the barium current approximately 4-fold. The largest contributor to the increase in barium current with bFGF treatment is a 6-fold increase in the high threshold voltage activated omega-conotoxin sensitive barium current. Smaller increases in current produced by bFGF treatment of PC12 cells are observed for the dihydropyridine sensitive and dihydropyridine/conotoxin insensitive currents. The bFGF-induced increases in barium currents are dependent on tyrosine phosphorylation, since the effects of bFGF are blocked by genistein, a tyrosine kinase inhibitor. This system will ultimately be useful in understanding the signaling pathways that control calcium channel expression in response to growth factors.

The small conductance calcium-activated potassium channel regulates ion channel expression in C3H10T1/2 cells ectopically expressing the muscle regulatory factor MRF4.

We investigated small conductance (SK) potassium channel-mediated regulation of muscle-specific, ion channel functional expression in the C3H10T1/2-MRF4 cell model system, a stable fibroblast line ectopically overexpressing the myogenic regulatory transcription factor, MRF4. Mitogenic stimulation of C3H10T1/2-MRF4 cells with basic fibroblast growth factor negatively regulates MRF4 transcriptional activity, inhibiting myogenesis. Using patch clamp techniques we found that mitogenic stimulation of C3H10T1/2-MRF4 cells also up-regulated SK. SK is a charybdotoxin-sensitive, apamin-insensitive channel that exerts positive proliferative control in fibroblasts. Mitogen withdrawal, which removes negative regulation of MRF4 thus initiating myogenesis, also eliminated SK channel currents, coincident both with induction of acetylcholine receptor channels, and up-regulation of muscle inward rectifier potassium channels. Addition of the SK channel blocker charybdotoxin to growth factor-containing culture medium overcame basic fibroblast growth factor-induced negative regulation of MRF4, as evidenced by induction of inward rectifier potassium and acetylcholine receptor channel expression identical to that observed in mitogen-withdrawn cells. Thus, the SK channel can govern electrophysiological phenotype in C3H10T1/2-MRF4 cells, consistent with an ability of SK to affect MRF4-dependent transcriptional activity. SK appears to be a pivotal signaling component for growth factor regulation of both cell proliferation and differentiation.

Kinase C activator 1,2-oleoylacetylglycerol attenuates voltage-dependent calcium current in sensory neurons.

The diacylglycerol analogue 1,2-oleoylacetylglycerol (OAG) and the phorbol ester 12-deoxyphorbol 13-isobutyrate (DPB) were tested for their effects on the voltage-dependent calcium (Ca) current in embryonic chicken dorsal root ganglion neurons in vitro. OAG (0.6-60 microM) and DPB (0.01-50 microM) produced reversible decreases in Ca current. Neither drug affected resting membrane conductance, the voltage-dependent potassium current, or the Ca current-voltage relationship. The concentrations of OAG and DPB that reduced Ca current correlate well with those concentrations that have been shown, in other systems, to activate protein kinase C-dependent phosphorylation. The time course for OAG action on Ca current is also consistent with an involvement of kinase C. Incubation of dorsal root ganglion cells in 60 microM OAG prevented further reductions in Ca current by either 50 microM DPB or 10 microM norepinephrine, a known modulator of the voltage-dependent Ca channel in these cells. This evidence suggests that protein kinase C may play a role in modulating Ca channel function.

Potassium channel induction by the Ras/Raf signal transduction cascade.

p21ras plays a critical role in cell growth, differentiation, and oncogenic transformation. However, the final physiological effectors of p21ras-mediated signal transduction remain to be determined. We have used patch clamp electrophysiology, pharmacological agents, and transfection with specific Ras or Raf plasmids, to demonstrate that induction of a unique Ca(2+)-activated K+ channel in murine fibroblast cell lines depends on p21ras and its immediate downstream target, the Raf kinase. The importance of this channel in mitogenic signaling is further indicated by its induction in nontransformed cells by epidermal growth factor and platelet-derived growth factor and the ability of K+ channel blockers to inhibit cell proliferation. We suggest that this Ca(2+)-activated K+ channel is one ultimate physiological target of p21ras-mediated signal transduction and that it may play a role in cell proliferation and ras transformation.

p21ras signaling is necessary but not sufficient to mediate neurotrophin induction of calcium channels in PC12 cells.

Nerve growth factor and basic fibroblast growth factor bind to and activate receptor tyrosine kinases, causing sequential signaling via the p21ras/extracellular signal-regulated kinase pathway. The necessity and sufficiency of this signaling pathway in transducing neuronal differentiation have been tested in the PC12 cell model. Although necessary for morphological changes, the sufficiency of p21ras-mediated signaling in these events has come into question. We report that growth factor induction of voltage-gated calcium channels, a hallmark of physiological differentiation, also requires p21ras-mediated signaling, but cannot be driven by p21ras activation alone. Thus, constitutive expression of the dominant negative N17ras mutant blocks growth factor-induced increases in Omega-conotoxin GVIA-sensitive, nimodipine-sensitive, and Omega-conotoxin GVIA/nimodipine-resistant calcium currents, but it does not block sodium current induction. However, manipulations that produce sustained activation of the p21ras signaling pathway and the neurite extension characteristic of morphological differentiation fail to increase calcium channel current densities. These results indicate the existence of distinct signaling requirements for morphological and physiological differentiation and further emphasize the importance of p21ras-independent signaling pathways in growth factor-mediated neuronal development.