SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes

Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane-directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP-2/synaptobrevin-2, syntaxin-2 and -4, nsec-1/munc-18-1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP-2, the small GTP-binding protein Rab3a, and the target SNARE syntaxin-4 were up-regulated. VAMP-2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network; in situ anti-VAMP-2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane-directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.     

Thrombospondin 1 and thrombospondin 2 are expressed as both homo- and heterotrimers.

There exist two distinct thrombospondin molecules (designated TSP1 and TSP2) which are encoded by separate genes. TSP1 is a trimeric cell surface and extracellular matrix molecule. Sequence comparison reveals that the 2 cysteines involved in interchain disulfide linkage and trimer assembly in TSP1 are conserved in TSP2 (Laherty, C. D., O'Rourke, K., Wolf, F. W., Katz, R., Seldin, M. F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 3274-3281). Swiss 3T3 fibroblasts express both TSP1 and TSP2, and, therefore, an important question is whether TSP in such cells is expressed as homotrimers or as heterotrimers. We find that Swiss 3T3 cells and epithelial cells transfected with TSP expression vectors express both homo- and heterotrimeric forms of TSP. In addition, homotrimeric TSP2 has a lower affinity for heparin than homotrimeric TSP1. Thus, the heparin affinity of TSP can be modulated by the expression of TSP as homo- or heterotrimers.     

Hu neuronal proteins are expressed in proliferating neurogenic cells

We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1–2 h) of the DNA precursor analog bromodeoxyruridine (BrdU) in conjunction with MAb 16A11, we observed Hu⁺/BrU⁺ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu⁺ cells were ambiguously BrdU⁺ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation. 1994 John Wiley & Sons, Inc.     

Glucocorticoid effects on newly synthesized proteins in muscle and non-muscle cells cultured from neonatal rat hearts

To analyze direct effects of steroids on the rates of synthesis (and/or degradation) of newly synthesized proteins of the rat heart, we have used high resolution two-dimensional gel electrophoresis and autoradiography. A collective steroid domain of nineteen proteins, comprising fifteen with an increased rate of synthesis and four with a decreased rate of synthesis, was consistently seen in cultures of cardiac muscle and non-muscle cells from neonatal rats following 24 h incubation with 10(-7) dexamethasone. Similarly, incubation with 10(-7) M sex steroids, mineralocorticoids, and other glucocorticoids including the highly selective compound RU26988, established the glucocorticoid-specificity of the response. Different subsets of this glucocorticoid domain were seen for collagenase- or trypsin-dispersed primary cultures of cardiac muscle and non-muscle cells or for passaged cultures of cardiac non-muscle cells. Six polypeptides were consistently induced in all cardiac cultures, regardless of cell morphology. Two polypeptides were consistently induced only in those cultures containing cardiac non-muscle cells, whereas protein l, of identical Mr(approximately 52K) and pI (approximately 5.3) to desmin, was induced only in cultures of spontaneously contractile cardiac muscle cells. The glucocorticoid domain proteins described herein represent direct steroid effects on cardiac cells and are therefore candidate mediators of physiological glucocorticoid effects on, for example, differentiation and contractility.     

Multiple growth hormone-binding proteins are expressed on insulin-producing cells

The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as disulfide-linked multimers of Mr 270-350K (300K). In addition, a minor Mr 180K GH-binding protein is identified which does not appear to be associated with other proteins by disulfide bridges. A plasma membrane-enriched fraction accounts for 86% of the RIN-cell GH-binding activity while cytosol and intracellular organelles are low in GH-binding activity. The plasma membrane-bound activity is soluble in Triton X-100 with intact hormone binding characteristics. The apparent KD in detergent solution is estimated to 18 ng/ml (8 x 10(-10) M). 125I-hGH-affinity cross-linking to intact and detergent-solubilized membranes as well as hGH-affinity purified protein reveals labeled proteins of Mr 180K and Mr 285-350K. In contrast to the cross-linked Mr 300K complexes of intact cells those of disintegrated cellular material are resistant to reduction with dithiothreitol, and it is speculated that this is due to intersubunit cross-linking of the disulfide-linked Mr 110K GH-binding subunits. The GH-binding proteins are purified approximately 100-fold by one cycle of hGH-affinity chromatography and five major proteins of Mr 180K, 94K, 86K, 64K, and 54K are identified by silver staining in the purified fraction. It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production.     

Distribution of actin and myosin in muscle and non-muscle cells

Summary Specific anti-actin and anti-myosin antibodies were shown to react in single and double immunofluorescence sandwich tests with identical sites in non-muscle cells in frozen sections of tissues and in cultured cells. In tissues, both antibodies reacted with liver cell membranes, parts of renal glomeruli, brush borders and peritubular fibrils of renal tubules, brain synaptic junctions, and membranes of lymphoid cells in thymic medulla, lymph nodes and spleen. Both antibodies reacted strongly with long parallel cytoplasmic fibrils in cultured fibroblasts, and with disrupted fibrils in cytochalasin-B treated cells. In neuroblastoma cells both antibodies gave prominent staining of growth cones and microspikes. The observation that the distribution of myosin parallels that of actin in non-muscle cells argues strongly in favour of a functional interaction between the two molecules in the generation of contractile activity in nonmuscle cells.The authors thank Dr. M. Owen, National Institute of Medical Research, Mill Hill, for the gift of rabbit anti-actin antibodyOn sabbatical leave from Monash University, and supported by a Commonwealth Medical FellowshipThe Brompton Hospital, London     

The LIM proteins FHL1 and FHL3 are expressed differently in skeletal muscle

We have determined the complete mRNA sequence of FHL3 (formerly SLIM2). We have confirmed that it is a member of the family of LIM proteins that share a similar secondary protein structure, renamed as Four-and-a-Half-LIM domain (or FHL) proteins in accordance with this structure. The "half-LIM" domain is a single zinc finger domain that may represent a subfamily of LIM domains and defines this particular family of LIM proteins. The distribution of FHL mRNA expression within a variety of murine tissues is complex. Both FHL1 and FHL3 were expressed in a number of skeletal muscles while FHL2 was expressed at high levels in cardiac muscle. Localisation of FHL3 to human chromosome 1 placed this gene in the proximity of, but not overlapping with, alleles associated with muscle diseases. FHL1 and FHL3 mRNAs were reciprocally expressed in the murine C2C12 skeletal muscle cell line and this suggested that the pattern of expression was linked to key events in myogenesis.     

Non-identity of muscle and non-muscle actins.

Tryptic peptide maps of actin prepared from chicken muscle and chick brain appear to be very similar, but not identical. Brain actin lacks at least one peptide found in muscle actin and its fingerprint contains approximately six additional peptides. Whether these differences between muscle and cytoplasmic actins are due to their synthesis from different genes or to post-translational modification is not yet known.     

Exercise and CaMK activation both increase the binding of MEF2A to the Glut4 promoter in skeletal muscle in vivo

In vitro binding assays have indicated that the exercise-induced increase in muscle GLUT4 is preceded by increased binding of myocyte enhancer factor 2A (MEF2A) to its cis-element on the Glut4 promoter. Because in vivo binding conditions are often not adequately recreated in vitro, we measured the amount of MEF2A that was bound to the Glut4 promoter in rat triceps after an acute swimming exercise in vivo, using chromatin immunoprecipitation (ChIP) assays. Bound MEF2A was undetectable in nonexercised controls or at 24 h postexercise but was significantly elevated approximately 6 h postexercise. Interestingly, the increase in bound MEF2A was preceded by an increase in autonomous activity of calcium/calmodulin-dependent protein kinase (CaMK) II in the same muscle. To determine if CaMK signaling mediates MEF2A/DNA associations in vivo, we performed ChIP assays on C(2)C(12) myotubes expressing constitutively active (CA) or dominant negative (DN) CaMK IV proteins. We found that approximately 75% more MEF2A was bound to the Glut4 promoter in CA compared with DN CaMK IV-expressing cells. GLUT4 protein increased approximately 70% 24 h after exercise but was unchanged by overexpression of CA CaMK IV in myotubes. These results confirm that exercise increases the binding of MEF2A to the Glut4 promoter in vivo and provides evidence that CaMK signaling is involved in this interaction.     

Dynamics of the endoplasmic reticulum in living non-muscle and muscle cells

The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3'-dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non-muscle PtK2 cell: 1) a lacy network of irregular polygons and 2) long strands of ER that are found aligned along stress fibers. In cross-striated myotubes there was a periodic localization of fluorescence over each I-band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the alignment of the long strands of ER alon stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123-stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.     

A chemical comparison of tropomyosins from muscle and non-muscle tissues.

Tropomyosins from six different calf tissues: aorta (smooth muscle), skeletal muscle, heart, brain, pancreas and platelets have been isolated, as well as a tropomyosin from mouse fibroblasts. The three muscle tropomyosins have identical polypeptide molecular weights (35,000), paracrystal periodicity and fine structure, and very similar peptide maps. The four non-muscle tropomyosins also have identical polypeptide molecular weights (30,000), paracrystal periodicity and fine structure, and very similar peptide maps. All tropomyosins examined have the same C-terminal amino acid, isoleucine and a blocked N terminal. These findings indicate that muscle and non-muscle tropomyosins are grouped into two similar but non-identical classes of protein. The two classes have at least ten peptide differences out of 31 total peptides, each group having several peptides not found in the other group. This suggests that the two classes of tropomyosins are coded for by different gene classes. It is likely that both gene classes evolved from an ancestral gene by a process involving gene duplication.Peptide maps of skeletal muscle tropomyosins from rabbit, calf and chick, and of non-muscle tropomyosins from rabbit, mouse and calf show few species differences. This suggests that tropomyosin is a highly conserved molecule.     

The MEF2A and MEF2D function as scaffold proteins that interact with HDAC1 or p300 in SOD3 expression in THP-1 cells

Superoxide dismutase 3 (SOD3) is a SOD isozyme and plays a key role in extracellular redox homeostasis. We previously demonstrated that histone acetylation is involved in 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited SOD3 expression in human monocytic THP-1 cells; however, the molecular mechanisms responsible for its expression have not yet been elucidated in detail. The results of the present study demonstrated that the binding of histone deacetylase 1 (HDAC1) to the SOD3 promoter region contributed to SOD3 silencing in basal THP-1 cells. On the other hand, the dissociation of HDAC1 from the SOD3 promoter region and the enrichment of p300, a histone acetyltransferase (HAT), within that region were observed in TPA-induced THP-1 cells. Myocyte enhancer factor 2 (MEF2) functions as a scaffold protein that interacts with histone deacetylases (HDAC) or HAT and regulates gene expression. The present results showed that the MEF2A and MEF2D function as mediators for TPA-elicited SOD3 expression by interacting with HDAC or p300. Additionally, the knockdown of MEF2A or MEF2D in human skin fibroblasts suppressed SOD3 expression at the mRNA and protein levels. Our results provide an insight into epigenetic regulation of redox gene expression, and may ultimately contribute to suppressing the progression of tumours and vascular diseases.     

VAMP2 is expressed in muscle satellite cells and up-regulated during muscle regeneration

Membrane trafficking is one of the most important mechanisms involved in the establishment and maintenance of the forms and functions of the cell. However, it is poorly understood in skeletal muscle cells. In this study, we have focused on vesicle-associated membrane proteins (VAMPs), which are components of the vesicle docking and fusion complex, and have performed immunostaining to investigate the expression of VAMPs in rat skeletal muscle tissue. We have found that VAMP2, but not VAMP1 or VAMP3, is expressed in satellite cells. VAMP2 is also expressed in myofibers in the soleus muscle and nerve endings. This is consistent with previous studies in which VAMP2 has been shown to regulate GLUT4 trafficking in slow-twitch myofibers in soleus muscle and neurotransmitter release in nerve endings. As satellite cells are quiescent myogenic cells, the expression of VAMP2 has further been examined in regenerating muscles after injury by the snake venom, cardiotoxin; we have observed enhanced expression of VAMP2 in immature myotubes with a peak at 3 days after injury. Our findings suggest that VAMP2 plays roles in quiescent satellite cells and is involved in muscle regeneration. The nature of the material transported in the VAMP2-bearing vesicles in satellite cells and myotubes is still under investigation. This work was supported by a research grant (17A-10) for nervous and mental disorders from the Ministry of Health, Labor, and Welfare of Japan, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.