Rodrigo Bravo

Selective expression of Jun proteins following axotomy and axonal transport block in peripheral nerves in the rat : evidence for a role in the regeneration process

Expression of the protein products of the immediate-early genes (IEGs), members of the fos, jun and krox families (Jun, Fos, and Krox, resp.) was investigated in the spinal cord and sensory ganglia (DRG) of normal rats; and following transection of, block of axonal transport in, or electrical stimulation of their peripheral axons. The nuclei of many moto- and DRG neurons showed a faint basal immunoreactivity (IR) for Jun proteins, but not for Fos or Krox proteins. There was a strong and selective induction of Jun-IR in moto- and DRG neurons after peripheral nerve transection or crush, or colchicine- or vinblastine-induced block of axonal transport. The Jun-IR induced by nerve transection disappeared after nerve regeneration. In contrast, Jun, Fos and Krox proteins were all induced transynaptically in spinal dorsal horn neurons following electrical stimulation of the C-fibers in the afferent nerves. Thus in differentiated neurons in vivo these IEG proteins can be expressed either independently or concomitantly depending on the type of stimulus.

Expression of immediate early gene proteins following axotomy and inhibition of axonal transport in the rat central nervous system.

The expression of the immediate early gene-encoded proteins c-Jun, Jun B, Jun D, c-Fos, Fos B and Krox-24 in central neurons following transection of, or inhibition of, axonal transport in their axons was investigated in the rat using immunocytochemistry. Transection of the medial forebrain bundle, which produces an essentially complete axotomy of neurons in the ipsilateral mammillary nucleus, substantia nigra pars compacta, ventral tegmental area and parafascicularis, induced the expression of c-Jun, Jun D and, to a lesser extent, Krox-24, in these nuclei. Microinjection of colchicine into the medial forebrain bundle to chemically inhibit axonal transport similarly induced the expression of these proteins in these areas. The expression of the proteins was first evident 24 h after transection, reached a maximum at 48 h and was still present after 10 days. However, after 30 days the proteins were absent from the substantia nigra, ventral tegmentum and parafascicularis, and were still present only in the mammillary nuclei. The other immediate early genes, Jun B, c-Fos and Fos B, were never expressed above the basal levels seen in untreated rats. Transection of the corpus callosum and the hippocampal commissure, which produces only a partial axotomy of neurons in the cerebral cortex and hippocampus, respectively, did not induce the expression of any of the genes in these neurons. Microinjection of colchicine or vinblastine to produce a localized inhibition of axonal transport in the cerebral cortex, hippocampus, thalamus and cerebellum also induced the expression of c-Jun, Jun D and, again to a lesser extent, Krox-24, in neurons surrounding the injection site. In contrast to this selective expression, administration of the neuronal excitant metrazole induced the expression of all six immediate early gene proteins in central nervous system neurons. These results demonstrate that transection of, or inhibition of, transport in the axons of central neurons induces a particular pattern of expression of transcriptionally operating immediate early genes that may be related to the regenerative competency of the neurons.

The KROX-24 protein, a new transcription regulating factor : expression in the rat central nervous system following afferent somatosensory stimulation

The expression of the protein product encoded by the Krox-24 gene was investigated immunocytochemically in the central nervous system of adult rats. Immunoreactivity (IR) of the KROX-24 protein which is a nuclear transacting transcription factor, showed a pattern of nuclear staining. Basal KROX-24-IR was visible in the superficial layers of spinal dorsal horn and trigeminal nucleus, and in many areas of the brain including the cerebellum, nucleus raphe magnus, colliculi, periaqueductal gray, hypothalamus, geniculate nuclei, caudate putamen, amygdala, hippocampus, lateral septal nucleus, olfactory tubercle and cerebral cortex. Electrical stimulation of sciatic nerve A- and C-fibers, but not of A alpha- and A beta-fibers alone, induced transient expression of KROX-24 in numerous spinal neurons in the termination area of the stimulated nerve. One hour following the onset of this stimulation of the sciatic nerve the distribution of KROX-24-IR was investigated in the brain and compared to untreated rats. In stimulated animals, the KROX-24-IR was induced in many areas including the lateral reticular nucleus, parabrachial nucleus, periaqueductal gray, hypothalamus, amygdala and lateral habenular nucleus.

Increased Brain Transcription Factor Expression by Angiotensin in Genetic Hypertension

A stimulated brain renin-angiotensin system has been implicated in genetic hypertension. We compared the effects of an intracerebroventricular injection of angiotensin II (100 ng) on the expression of inducible transcription factors c-Fos, c-Jun, and Krox-24 in the brain of spontaneously hypertensive rats (SHR). in Wistar rats with nephrogenic hypertension induced by aortic banding, and in normotensive Wistar-Kyoto and Wistar rats immunohistochemically. Generally, the angiotensin II-induced transcription factor expression was strictly confined to four distinct forebrain areas: the subfornical organ, median preoptic area, paraventricular nucleus, and supraoptic nucleus. In SHR, the angiotensin II-induced c-Fos and c-Jun expressions were significantly enhanced compared with those in normotensive control strains as well as in secondary hypertensive Wistar rats. Krox-24 expression in the subfornical organ, median preoptic area, and paraventricular nucleus of SHR was also significantly increased compared with that in all control strains. In the supraoptic nucleus, significant differences could be discriminated between SHR and secondary hypertensive Wistar rats. Injection of isotonic saline or arginine vasopressin (100 ng) as controls did not induce any expression of c-Fos, c-Jun, or Krox-24. Our findings demonstrate an enhanced sensitivity of SHR to angiotensin II-induced transcription factor expression in distinct brain areas involved in central blood pressure and osmotic control that is independent of blood pressure.

Induction of c-Jun and Suppression of CREB Transcription Factor Proteins in Axotomized Neurons of Substantia Nigra and Covariation with Tyrosine Hydroxylase

In adult rats, the expression of transcription factor proteins c-Jun and CREB and their colocalization with tyrosine hydroxylase (TH) were investigated in neurons of the substantia nigra compacta (SNC) axotomized by stereotaxic unilateral transection of the medial forebrain bundle (MFB). Axotomized SNC neurons were identified by injection of the retrograde tracer horseradish-peroxidase-coupled-gold (HRP-gold) into the ipsilateral striatum 5 days prior to MFB transection. Nuclear c-Jun immunoreactivity (IR) appeared 36 h after MFB transection in SNC neurons, was maximal after 5 days, and declined after 10 days. c-Jun-IR was visible in HRP-gold-labeled SNC neurons, demonstrating that c-Jun is in fact expressed in axotomized neurons. The constitutively expressed CREB (calcium/cAMP response element-binding protein, syn. CREB-1) was present in apparently all neuronal and glial cells in the brains of untreated rats including those SNC neurons that coexpressed TH. Three days following MFB transection, the nuclear CREB-IR disappeared in the axotomized SNC neurons labeled by TH-IR and was almost completely absent after 20 days in this neuronal population. The TH-IR rapidly declined 5 days after MFB transection, and 10 and 100 days post-axotomy the number of TH-labeled neurons was reduced by 52 and 80%, respectively. During this period, the majority of surviving TH positive neurons coexpressed c-Jun but were immunonegative for CREB. Between 3 and 60 days following MFB transection, the number of CREB-labeled glial cell nuclei increased in the ipsilateral substantia nigra by about 80%. Concomitantly, expression of GFAP, a marker protein for astrocytes, was also enhanced whereas nuclear c-Jun-, JunD-, and c-Fos-IR did not change in glial cells. These findings demonstrate that c-Jun can be expressed in axotomized neurons during the absence of CREB and suggest a role of c-Jun in the transcriptional control of the TH gene.

Immediate early gene responses of NIH 3T3 fibroblasts and NMuMG epithelial cells to TGF beta-1.

Transforming growth factor beta has a wide range of physiological effects on cell growth and metabolism. We have previously reported on the rapid induction of jun transcription factors in TGF beta-treated cells. Here we show that the early genomic response to TGF beta-1 includes activation of a broad spectrum of serum-inducible genes both in NIH 3T3 fibroblasts and in NMuMG epithelial cells, which are growth-stimulated and growth-inhibited by TGF beta, respectively. Of particular interest is the presence of a putative nuclear DNA-binding receptor (N10) and zinc finger transcription factors (Krox 20 and Krox 24) among the TGF beta-induced genes. In addition to the stimulatory effects of TGF beta, expression of a few genes including c-myc is decreased in both types of cells. In cells transformed by neu or ras oncogenes the immediate early mRNA responses to TGF beta are deregulated. Our results suggest that certain transcription factors are required for both positive and negative regulation of cell proliferation by TGF beta, and that their relative concentrations may determine the subsequent cellular responses.