The immunophilin ligand FK506, but not the P38 kinase inhibitor SB203580, improves function of adult rat muscle reinnervated from transplants of embryonic neurons.

Injury to the adult CNS often involves death of motoneurons, resulting in the paralysis and progressive atrophy of muscle. There is no effective therapy to replace motoneurons in the CNS. Our strategy to replace neurons and to rescue denervated muscles is to transplant dissociated embryonic day 14-15 (E14-15) ventral spinal cord cells into the distal stump of a peripheral nerve near the denervated muscles. Here, we test whether long-term delivery of two pharmacological inhibitors to denervated muscle, FK506 or SB203580, enhances reinnervation of muscle from embryonic cells transplanted in the tibial nerve of adult Fischer rats. FK506, SB203580 (2.5 mg/kg) or saline was delivered under the fascia of the medial gastrocnemius muscle for 4 weeks, beginning when muscles were denervated by section of the sciatic nerve. After 1 week of nerve degeneration, one million E14-15 ventral spinal cord cells were transplanted into the distal tibial nerve stump of each rat in the three treatment groups. Ten weeks later, all cell transplants had neuron-specific nuclear protein (NeuN) positive neurons. Neuron survival and axon regeneration were similar across treatments. An average (+/-S.E.) of 210+/-66, 100+/-36 and 176+/-58 myelinated axons grew distally from the cell transplants of rats with muscles treated with FK506, SB203580 or saline, respectively. Regenerating axons in muscles of all three treatments groups were detected with antibodies against phosphorylated neurofilaments and synaptophysin, and motor end plates were labeled with alpha-bungarotoxin. Muscles of rats that received transplants of media only had no axon growth, indicating that the muscles were denervated. The mean muscle fiber areas of rats that received cell transplants and had long-term delivery of FK506, SB203580 or saline to muscles were significantly larger than those of denervated muscle fibers. Thus, cell transplantation reduced muscle atrophy. Transplantation of embryonic cells also resulted in functional muscle reinnervation. Electromyographic activity and force were evoked from >90% of the muscles of rats with cell transplants, but not from denervated muscles. FK506-treated muscles were significantly more fatigue resistant than naive control muscles. FK506-treated muscles also had significantly stronger motor units than those in SB203580 or saline-treated muscles. These data suggest that a pathway regulated by FK506 improves the function of muscles reinnervated by embryonic neurons placed in peripheral nerve.

Identification of a point mutation in the thyrotropin receptor of the hyt/hyt hypothyroid mouse.

The hyt/hyt hypothyroid mouse has an autosomal recessive, fetal-onset, severe hypothyroidism related to TSH hyporesponsiveness and associated with elevated TSH. Our previous work has suggested that the hypothyroidism and TSH hyporesponsiveness may result from a mutation in the hyt/hyt TSH receptor (TSHr) of the thyroid gland. Based on DNA sequencing of the entire coding region of the TSHr gene from the wild-type BALB/cBY +/+ mouse, the +/+ TSHr is 92% and 94% identical at the nucleotide and amino acid residue levels, respectively, compared to the rat TSHr gene. The coding region of the hyt/hyt TSHr, compared to that of the +/+ TSHr, has a single base change, CCG to CTG, at nucleotide position 1666, which leads to the replacement of a highly conserved proline at amino acid position 556 with a leucine in transmembrane domain IV. This mutation was introduced by site-directed mutagenesis into the wild-type human TSHr and transiently expressed in COS-7 cells. Although the size and abundance of the mutant TSHr mRNA suggested that there was no effect on the nature of the mRNA, TSH binding and the response to TSH in transfected cells were abolished. Further studies are necessary to clarify how the Pro to Leu replacement interferes with receptor expression on the cell surface or influences TSH binding. These functional consequences of the mutation appear to account for the observed TSH hyporesponsiveness and hypothyroidism in the hyt/hyt mouse.

Absence of androgen-mediated transcriptional effects in osteoblastic cells despite presence of androgen receptors.

Androgen excess and deficiency affect skeletal maturation and bone cell function. Understanding the molecular basis for these androgen effects could improve therapy/prevention of short stature and osteoporosis. Androgens act through binding to androgen receptors (ARs), which modulate gene transcription via interactions with DNA response elements on target genes. Because osteoblasts contain ARs at levels just below certain androgen-sensitive tissues, we sought to define the function of AR in a number of commonly used osteoblastic cell lines. Presence and quantification of AR protein and mRNA were evaluated by ligand binding assay, western blotting, and RNAse protection assay. AR-containing osteoblastic cell lines were exposed to nonaromatizable androgens and effects on gene expression were assessed. We found no evidence for direct effects of androgen on endogenous genes nor was androgen involved in modulation of parathyroid hormone effects on early gene activation. Androgen-sensitive reporter gene constructs were stimulated by androgen only when AR cDNA expression vectors were introduced into cells by cotransfection. We conclude that, in commonly used osteoblastic cell lines, the presence of AR at the levels described here does not guarantee androgen transcriptional activity. The effects of androgen on bone in vivo may involve direct stimulation of osteoblastic cells in a different setting or stage of differentiation. Alternatively, androgen may act on bone cells other than osteoblasts, or through metabolic conversion to estrogens.

Hepatocyte growth factor and its actions in growth plate chondrocytes.

Hepatocyte growth factor (HGF) has been implicated as a paracrine regulator of organogenesis and repair in many tissues. Here we have studied the expression and actions of HGF in intact rachitic rat growth plate and derived cultures of proliferative zone chondrocytes. In vivo and in vitro chondrocytes express HGF mRNA; 1,25(OH)2 has a three-fold maximal stimulatory effect, which can be blocked by H-7, an inhibitor of protein kinase C. Although HGF elaboration and action generally follow a paracrine model, chondrocytes appear capable of both expressing and responding to HGF. mRNA encoding the HGF receptor (c-met) was detected in both growth cartilage and derived chondrocyte cultures. HGF addition to chondrocyte cultures increased collagen II mRNA and alkaline phosphatase enzymatic activity to degrees comparable to that observed for active vitamin D metabolites. Combining HGF and 1,25-D evoked a synergistic response (ninefold) of alkaline phosphatase activity. To assess whether a similar stimulatory effect might be seen with bioactive peptides and HGF, we investigated the effect of HGF pretreatment on acute responses of chondrocytes to synthetic human calcitonin, an anabolic chondrocyte regulator whose skeletal action are mediated principally by cAMP elevation and subsequent protein kinase A activation. CT's maximal activation of protein kinase A was increased by prior HGF treatment from 56% to 78%. In concert, our findings indicate that in addition to HGF's classical paracrine role during skeletal growth, this growth factor may modulate hormonal sensitivity of the chondrocyte during proliferation, differentiation, and/or apoptosis.

Knee joint immobilization decreases aggrecan gene expression in the meniscus.

Aggrecan is the major proteoglycan of the meniscus, and its primary function is to give the meniscus its viscoelastic compressive properties. The objective of this study was to determine the effect of joint immobilization on aggrecan gene expression in the meniscus. The right hindlimbs of six mature beagles were knee cast-immobilized in 90 degrees of flexion and supported by a sling to prevent weightbearing, while the contralateral limb was left free to bear weight. The animals were sacrificed at 4 weeks, and the anterior and posterior halves of the medial and lateral menisci were analyzed separately. Analysis of aggrecan gene expression by quantitative polymerase chain reaction showed decreased aggrecan gene expression in menisci from immobilized knees (P < 0.01, two-way analysis of variance). Aggrecan gene expression decreased by a factor of 2 to 5.5 in the different regions examined. Analysis of the composition of the meniscus also showed decreased proteoglycan content and increased water content with immobilization (P < 0.05, two-way analysis of variance). These results show that joint immobilization can significantly affect meniscal cellular activity and composition and can therefore potentially affect meniscal function.

Cartilage metalloproteases in disuse atrophy.

A canine knee model of disuse atrophy produced by nonrigid fixation (sling) was characterized in respect to variables of proteoglycan size distribution, as well as biomechanical properties versus controls. Using this model, we found, in addition to the accepted dogma attributing changes to reduced protein synthesis by chondrocytes, that there is elevation of proteases and depression of tissue inhibitor of metalloproteases (TIMP) in atrophic knee cartilage. The findings are suggestive of cartilage remodelling reminiscent of bone remodelling in disuse atrophy reported by others. Whether the abnormal changes of protease-TIMP balance in knee cartilage can be retarded prophylactically by concurrent treatment with pentosan polysulfate and insulin like growth factor 1 remains uncertain.

Embryonic cord transplants in peripheral nerve restore skeletal muscle function.

The rapid atrophy of skeletal muscle after denervation severely compromises efforts to restore muscle function. We have transplanted embryonic day 14-15 (E14-E15) ventral spinal cord cells into adult Fischer rat tibial nerve stump to provide neurons for reinnervation. Our aim was to evaluate medial gastrocnemius reinnervation physiologically because this transplant strategy will only be effective if the reinnervated muscle contracts, generates sufficient force to induce joint movement, and is fatigue resistant enough to shorten repeatedly. Twelve weeks posttransplantation, brief duration electrical stimuli applied to the transplants induced medial gastrocnemius contractions that were strong enough to produce ankle movement in 4 of 12 rats (33%). The force of these four "low-threshold" reinnervated muscles and control muscles declined only gradually during five hours of intermittent, supramaximal stimulation and without depression of EMG potential area, which is strong evidence of functional neuromuscular junctions and fatigue resistant muscles. Sectioning of the medial gastrocnemius nerves confirmed that these contractions were innervation dependent. Weakness in low-threshold reinnervated muscles (8% control force) related to incomplete reinnervation, reductions in muscle fiber size, specific tension, and/or the presence of nonfunctional neuromuscular junctions. Muscle reinnervation achieved using this novel transplantation strategy may salvage completely denervated muscle and may provide the potential to evoke limb movement when injury or disease precludes or delays peripheral axon regeneration.

Regulation of rat interstitial collagenase gene expression in growth cartilage and chondrocytes by vitamin D3, interleukin-1 beta, and okadaic acid.

The interstitial collagenase produced by the rat growth plate chondrocytes is the homologue of the human collagenase-3, or matrix metalloproteinase-13. This enzyme is responsible for the loss of collagen during hypertrophy of chondrocytes and for the degradation of transverse septa in long bone growth. Rachitic rats (42 days, male Sprague-Dawley) had an 8-fold higher level of collagenase mRNA in the hypertrophic versus proliferative zone of growth plate cartilage. Intramuscular injection of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; 1.0 micrograms/kg body weight) in rachitic rats increased collagenase mRNA another 1.5-fold in the hypertrophic zone. The regulation of collagenase gene by 1,25-(OH)2D3 and interleukin (IL)-1 beta in cultured proliferative chondrocytes was studied by means of steady-state mRNA and half-life determination of mRNA using the transcriptional inhibitor actinomycin D, and nuclear run-on transcription analyses. Treatment of cells with 1,25-(OH)2D3 (10(-6) M) and IL-1 beta (2 ng/ml) increased collagenase mRNA 8- and 13-fold, respectively. Additionally, the collagenase mRNA half-life was increased by 1,25-(OH)2D3 and IL-1 beta. In the presence of a protein kinase C inhibitor, staurosporine, 1,25-(OH)2 D3 induction of collagenase mRNA was blocked. Here the addition of phorbol 12-myrisate 13-acetate (PMA) to activate protein kinase C increased collagenase mRNA 10-fold. However, in the presence of staurosporine (50 nM), PMA induction was blocked, whereas IL-1 beta was not. IL-1 beta is known to activate several phosphorylation pathways. Okadaic acid (500 nM), a protein phosphatase inhibitor, increased the relative collagenase mRNA abundance 10-fold. The rate of the rat collagenase gene transcription in nuclei was increased with 1,25-(OH)2D3, IL-1 beta and okadaic acid. In separate experiments, the collagenase promoter was ligated to a reporter plasmid and the plasmid was transfected into chondrocytes. The results showed that 1,25-(OH)2D3, IL-1 beta, and PMA increased reporter activity 2.5-, 2.8-, and 3.27-fold, respectively. Thus, there are multiple nuclear and cytoplasmic mechanisms by which cartilage modulators regulate rat interstitial collagenase gene expression.

Regulation of the rat interstitial collagenase promoter by IL-1 beta, c-Jun, and Ras-dependent signaling in growth plate chondrocytes.

In an attempt to better define molecular influences on rat interstitial collagenase gene expression in cartilage, the promoter function was characterized using transient transfection assay, electrophoresis mobility shift assay, and genetic analysis in isolated growth plate chondrocytes. Data from 5'-flanking deletion and selected mutations suggest that multiple cis elements in both the proximal and distal regions of the promoter were important in the regulation of promoter activity. A proximal tumor response element (TRE) was shown to be necessary for basal and interleukin (IL)-1 beta-inducible reporter gene activity. Cells stimulated by IL-1 beta (1 ng/ml; 18 h) had elevated TRE binding activity, and one of the factors involved was identified as the nuclear protein, c-Jun. Indeed, c-Jun directed antisense oligonucleotides reduced rat interstitial collagenase mRNA. A sense oligonucleotide was ineffective. Regulation of promoter activity was susceptible to Ras-dependent signaling as expression of dominant negative mutant of Ras kinase (pZIP-RasN17) reduced reporter gene activity. In a comparison of proximal promoter reporter plasmid activity between proliferative and hypertrophic cells, inhibition of Ras-dependent signaling was less effective in the later cell type. This study suggests that the activation of nuclear binding proteins that bind TRE may be a common event with IL-1 beta regulation. Moreover, these data suggest that the regulation of rat interstitial collagenase gene expression is a combinatorial process and multiple cis-acting regulatory sites may interact to exert different effects dependent on the stage of chondrocyte differentiation.