Estrogen and insulin-like-growth factor 1 (IGF-1) are potent mitogenic stimuli that share important properties in the control of cellular proliferation. However, the coupling between the signaling cascades of estrogen receptors ␣ and ␤ and the IGF-1 receptor (IGF-1R) is poorly understood. Therefore, we selectively transfected estrogen receptor ␣ or ␤ in COS7 and HEK293 cells, which contain IGF-1R. In presence of estrogen receptor ␣ but not ␤, 17␤-estradiol (E2) rapidly induces phosphorylation of the IGF-1R and the extracellular signal-regulated kinases 1/2. Furthermore, upon stimulation with E2, estrogen receptor ␣ but not ␤ bound rapidly to the IGF-1R in COS7 as well as L6 cells, which express all investigated receptors endogenously. Control experiments in the IGF-1R-deficient fibroblast cell line R ؊ showed that after stimulation with E2 only estrogen receptor ␣ bound to the transfected IGF-1R. Overexpression of dominant negative mitogen-activated protein kinases kinase inhibited this effect. Finally, estrogen receptor ␣ but not ␤ is required to induce the activation of the estrogen receptor-responsive reporter ERE-LUC in IGF-1-stimulated cells. Taken together, these data demonstrate that ligand bound estrogen receptor ␣ is required for rapid activation of the IGF-1R signaling cascade.Estrogen as well as insulin-like growth factor 1 (IGF-1) 1 are potent mitogens that are involved in a large array of processes that control proliferation and differentiation in mammalian cells (1, 2). Both mitogens act through receptor-mediated signaling pathways. The cross-talk between these two signaling pathways is currently under investigation (3-6). Estrogen is a steroid hormone that binds to members of the nuclear receptor superfamily (7), whereas IGF-1 as a peptide-growth factor binds to a transmembrane tyrosine kinase receptor, which signals via a series of phosphorylation events (2).Two different estrogen receptors, ER␣ and ER␤, which are encoded by genes located on different chromosomes, have been identified so far (8,9). Sequence analysis demonstrates a high degree of homology between ER␣ and ER␤ in the DNA-binding domain and the ligand-binding domain. However, there are significant differences in regions that would be expected to influence transcriptional activity. The ability of estrogen receptors to activate target gene transcription has been attributed to two regions: the N-terminal activation function 1 (AF-1) and the ligand-dependent AF-2, which is localized in the C-terminal hormone-binding domain (10, 11). AF-1 and AF-2 can activate transcription independently and synergistically, and they act in a promoter-and cell-specific manner (12, 13). Phosphorylation of a serine residue at position 118 is required for full action of the AF-1 (14). Both AF-1 and AF-2 are required to enhance transcription of target genes through AP-1 sites (15). Interestingly, ER␣ and ER␤ act differently at AP-1 sites (16), which may be due to differences in their AF domains (17). ER␣ and ER␤ can form homo-and heterodimers (18), and thus t...

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Sphingolipids containing 2-hydroxylated fatty acids are among the most abundant lipid components of the myelin sheath and therefore are thought to play an important role in formation and function of myelin. To prove this hypothesis, we generated mice lacking a functional fatty acid 2-hydroxylase (FA2H) gene. FA2H-deficient (FA2H Ϫ/Ϫ ) mice lacked 2-hydroxylated sphingolipids in the brain and in peripheral nerves. In contrast, nonhydroxylated galactosylceramide was increased in FA2H Ϫ/Ϫ mice. However, oligodendrocyte differentiation examined by in situ hybridization with cRNA probes for proteolipid protein and PDGF␣ receptor and the time course of myelin formation were not altered in FA2H Ϫ/Ϫ mice compared with wild-type littermates. Nerve conduction velocity measurements of sciatic nerves revealed no significant differences between FA2H Ϫ/Ϫ and wild-type mice. Moreover, myelin of FA2H Ϫ/Ϫ mice up to 5 months of age appeared normal at the ultrastructural level, in the CNS and peripheral nervous system. Myelin thickness and g-ratios were normal in FA2H Ϫ/Ϫ mice. Aged (18-month-old) FA2H Ϫ/Ϫ mice, however, exhibited scattered axonal and myelin sheath degeneration in the spinal cord and an even more pronounced loss of stainability of myelin sheaths in sciatic nerves. These results show that structurally and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids but that its long-term maintenance is strikingly impaired. Because axon degeneration appear to start rather early with respect to myelin degenerations, these lipids might be required for glial support of axon function.

The L-type calcium current (ICa,L) plays an important role in excitation-contraction coupling of heart cells, as it forms the major trigger for Ca¥-induced Ca¥ release from the sarcoplasmic reticulum (SR) and provides Ca¥ for refilling of the SR (Callewaert, 1992;Barry & Bridge, 1993). The voltage relation of this current is bell shaped with an inward current maximum at about 10 mV and a reversal potential around 60 mV (McDonald et al. 1994). At the top of the action potential between 40 and 50 mV, when ICa,L is activated, ICa,L is already near its reversal potential and thus small. Moreover, the potential of maximum ICa,L is not reached before the repolarization of the cell. The obvious contradiction that ICa,L is important for excitationcontraction coupling on the one hand, but is small during the action potential on the other hand, leads to the idea of applying voltage-clamp pulses consisting of digitized action potentials to record the time course of ICa,L directly during the action potential. This so-called action potential clamp 1. During an action potential the L-type Ca¥ current (ICa,L) activates rapidly, then partially declines leading to a sustained inward current during the plateau phase. The reason for the sustained part of ICa,L has been investigated here. 2. In the present study the mechanisms controlling the ICa,L during an action potential were investigated quantitatively in isolated guinea-pig ventricular myocytes by whole-cell patch clamp. To measure the actual time courses of ICa,L and the corresponding L-type channel inactivation (fAP) during an action potential, action potential-clamp protocols combined with square pulses were applied. 3. Within the first 10 ms of the action potential the ICa,L rapidly inactivated by about 50%; during the plateau phase inactivation proceeded to 95%. Later, during repolarization, the L_type channels recovered up to 25%. 4. The voltage-dependent component of inactivation during an action potential was determined from measurements of L-type current carried by monovalent cations. This component of inactivation proceeded rather slowly and contributed only a little to fAP. ICa,L during an action potential is thus mainly controlled by Ca¥-dependent inactivation. 5. In order to investigate the source of the Ca¥ controlling fAP, internal Ca¥ homeostasis was manipulated by the use of Ca¥ buffers (EGTA, BAPTA), by blocking Na¤-Ca¥ exchange, or by blocking Ca¥ release from the sarcoplasmic reticulum (SR). Internal BAPTA markedly reduced the L-type channel inactivation during the entire action potential, whereas EGTA affected fAP only during the middle and late plateau phases. Inhibition of Na¤-Ca¥ exchange markedly increased inactivation of L-type channels. Although blocking SR Ca¥ release decreased the fura_2-measured cytoplasmic Ca¥ concentration ([Ca¥]é) transient by about 90%, it reduced L-type channel inactivation only during the initial 50 ms of the action potential. Thus, it is Ca¥ entering the cell through the L-type channels that controls the inactivation proc...

Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive and sporadic forms of protein aggregation myopathies and cardiomyopathies. We generated R349P desmin knock-in mice, which harbor the ortholog of the most frequently occurring human desmin missense mutation R350P. These mice develop age-dependent desmin-positive protein aggregation pathology, skeletal muscle weakness, dilated cardiomyopathy, as well as cardiac arrhythmias and conduction defects. For the first time, we report the expression level and subcellular distribution of mutant versus wild-type desmin in our mouse model as well as in skeletal muscle specimens derived from human R350P desminopathies. Furthermore, we demonstrate that the missense-mutant desmin inflicts changes of the subcellular localization and turnover of desmin itself and of direct desmin-binding partners. Our findings unveil a novel principle of pathogenesis, in which not the presence of protein aggregates, but disruption of the extrasarcomeric intermediate filament network leads to increased mechanical vulnerability of muscle fibers. These structural defects elicited at the myofiber level finally impact the entire organ and subsequently cause myopathy and cardiomyopathy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1363-2) contains supplementary material, which is available to authorized users.