Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus.

Endochondral ossification is a developmental process by which cartilage is replaced by bone. Terminally differentiated hypertrophic chondrocytes are calcified, vascularized, and removed by chondroclasts before bone matrix is laid down by osteoblasts. In mammals, the malleus is one of three auditory ossicles that transmit vibrations of the tympanic membrane to the inner ear. The malleus is formed from a cartilaginous precursor without growth plate involvement, but little is known about how bones of this type undergo endochondral ossification. Here, we demonstrate that in the processus brevis of the malleus, clusters of osteoblasts surrounding the capillary loop produce bone matrix, causing the volume of the capillary lumen to decrease rapidly in post-weaning mice. Synchrotron X-ray tomographic microscopy revealed a concentric, cylindrical arrangement of osteocyte lacunae along capillaries, indicative of pericapillary bone formation. Moreover, we report that overexpression of Fosl1, which encodes a component of the AP-1 transcription factor complex, in osteoblasts significantly blocked malleal capillary narrowing. These data suggest that osteoblast/endothelial cell interactions control growth plate-free endochondral ossification through 'osteogenic capillaries' in a Fosl1-regulated manner.

Sema3a maintains normal heart rhythm through sympathetic innervation patterning.

Sympathetic innervation is critical for effective cardiac function. However, the developmental and regulatory mechanisms determining the density and patterning of cardiac sympathetic innervation remain unclear, as does the role of this innervation in arrhythmogenesis. Here we show that a neural chemorepellent, Sema3a, establishes cardiac sympathetic innervation patterning. Sema3a is abundantly expressed in the trabecular layer in early-stage embryos but is restricted to Purkinje fibers after birth, forming an epicardial-to-endocardial transmural sympathetic innervation patterning. Sema3a(-/-) mice lacked a cardiac sympathetic innervation gradient and exhibited stellate ganglia malformation, which led to marked sinus bradycardia due to sympathetic dysfunction. Cardiac-specific overexpression of Sema3a in transgenic mice (SemaTG) was associated with reduced sympathetic innervation and attenuation of the epicardial-to-endocardial innervation gradient. SemaTG mice demonstrated sudden death and susceptibility to ventricular tachycardia, due to catecholamine supersensitivity and prolongation of the action potential duration. We conclude that appropriate cardiac Sema3a expression is needed for sympathetic innervation patterning and is critical for heart rate control.

Forced expression of stabilized c-Fos in dendritic cells reduces cytokine production and immune responses in vivo.

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production by monocytic cells. We have shown that the transcription factor c-Fos is responsible for cAMP-mediated suppression of inflammatory cytokine production, and that c-Fos protein is stabilized by IKKß-mediated phosphorylation. We found that S308 is one of the major phosphorylation sites, and that the S308D mutation prolongs c-Fos halflife. To investigate the role of stabilized c-Fos protein in dendritic cells (DCs) in vivo, we generated CD11c-promoter-deriven c-FosS308D transgenic mice. As expected, bone marrow-derived DCs (BMDCs) from these Tg mice produced smaller amounts of inflammatory cytokines, including TNF-α, IL-12, and IL-23, but higher levels of IL-10, in response to LPS, than those from wild-type (Wt) mice. When T cells were co-cultured with BMDCs from Tg mice, production of Th1 and Th17 cytokines was reduced, although T cell proliferation was not affected. Tg mice demonstrated more resistance to experimental autoimmune encephalomyelitis (EAE) than did Wt mice. These data suggest that c-Fos in DCs plays a suppressive role in certain innate and adaptive immune responses.

Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis.

Bone homeostasis requires a delicate balance between the activities of bone-resorbing osteoclasts and bone-forming osteoblasts. Various molecules coordinate osteoclast function with that of osteoblasts; however, molecules that mediate osteoclast-osteoblast interactions by simultaneous signal transduction in both cell types have not yet been identified. Here we show that osteoclasts express the NFATc1 target gene Efnb2 (encoding ephrinB2), while osteoblasts express the receptor EphB4, along with other ephrin-Eph family members. Using gain- and loss-of-function experiments, we demonstrate that reverse signaling through ephrinB2 into osteoclast precursors suppresses osteoclast differentiation by inhibiting the osteoclastogenic c-Fos-NFATc1 cascade. In addition, forward signaling through EphB4 into osteoblasts enhances osteogenic differentiation, and overexpression of EphB4 in osteoblasts increases bone mass in transgenic mice. These data demonstrate that ephrin-Eph bidirectional signaling links two major molecular mechanisms for cell differentiation--one in osteoclasts and the other in osteoblasts--thereby maintaining bone homeostasis.

Fibroblast expression of an IκB dominant-negative transgene attenuates renal fibrosis.

It is not clear whether interstitial fibroblasts or tubular epithelial cells are primarily responsible for the profibrotic effects of NF-κB activation during renal fibrogenesis. Here, we crossed mice carrying a conditional IκB dominant-negative transgene (IκBdN) with mice transgenic for cell-specific FSP1.Cre (FSP1(+) fibroblasts) or γGT.Cre (proximal tubular epithelia) and challenged all progeny with unilateral ureteral obstruction. We determined NF-κB activation by nuclear localization of phosphorylated p65 ((p)p65) in renal tissues after 7 days. We observed inhibition of NF-κB activation in interstitial cells and tubular epithelia in obstructed kidneys of FSP1.Cre;IκBdN and γGT.Cre;IκBdN mice, respectively, compared with IκBdN controls (P < 0.05). Deposition of extracellular matrix, however, was significantly lower in the obstructed kidneys of FSP1.Cre;IκBdN mice but not in γGT.Cre;IκBdN mice (P < 0.05). In addition, levels of mRNA encoding the profibrotic PAI-1, fibronectin-EIIIA, and type I (α1) procollagen were significantly lower in obstructed kidneys of FSP1.Cre;IκBdN mice compared with γGT.Cre;IκBdN mice (P < 0.05). Taken together, these data support a profibrotic role for fibroblasts, but not proximal tubular epithelial cells, in modulating NF-κB activation during renal fibrogenesis.

Dominant negative suppression of Rad leads to QT prolongation and causes ventricular arrhythmias via modulation of L-type Ca2+ channels in the heart.

Disorders of L-type Ca2+ channels can cause severe cardiac arrhythmias. A subclass of small GTP-binding proteins, the RGK family, regulates L-type Ca2+ current (I(Ca,L)) in heterologous expression systems. Among these proteins, Rad (Ras associated with diabetes) is highly expressed in the heart, although its role in the heart remains unknown. Here we show that overexpression of dominant negative mutant Rad (S105N) led to an increase in I(Ca,L) and action potential prolongation via upregulation of L-type Ca2+ channel expression in the plasma membrane of guinea pig ventricular cardiomyocytes. To verify the in vivo physiological role of Rad in the heart, a mouse model of cardiac-specific Rad suppression was created by overexpressing S105N Rad, using the alpha-myosin heavy chain promoter. Microelectrode studies revealed that action potential duration was significantly prolonged with visible identification of a small plateau phase in S105N Rad transgenic mice, when compared with wild-type littermate mice. Telemetric electrocardiograms on unrestrained mice revealed that S105N Rad transgenic mice had significant QT prolongation and diverse arrhythmias such as sinus node dysfunction, atrioventricular block, and ventricular extrasystoles, whereas no arrhythmias were observed in wild-type mice. Furthermore, administration of epinephrine induced frequent ventricular extrasystoles and ventricular tachycardia in S105N Rad transgenic mice. This study provides novel evidence that the suppression of Rad activity in the heart can induce ventricular tachycardia, suggesting that the Rad-associated signaling pathway may play a role in arrhythmogenesis in diverse cardiac diseases.

Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression.

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.

Osteogenic Factor Runx2 Marks a Subset of Leptin Receptor-Positive Cells that Sit Atop the Bone Marrow Stromal Cell Hierarchy.

Bone marrow mesenchymal stem and progenitor cells (BM-MSPCs) maintain homeostasis of bone tissue by providing osteoblasts. Although several markers have been identified for labeling of MSPCs, these labeled cells still contain non-BM-MSPC populations. Studies have suggested that MSPCs are observed as leptin receptor (LepR)-positive cells, whereas osteoblasts can be classified as positive for Runx2, a master regulator for osteoblastogenesis. Here, we demonstrate, using Runx2-GFP reporter mice, that the LepR-labeled population contains Runx2-GFPlow sub-population, which possesses higher fibroblastic colony-forming units (CFUs) and mesensphere capacity, criteria for assessing stem cell activity, than the Runx2-GFP- population. In response to parathyroid hormone (PTH), a bone anabolic hormone, LepR+Runx2-GFPlow cells increase Runx2 expression and form multilayered structures near the bone surface. Subsequently, the multilayered cells express Osterix and Type I collagen α, resulting in generation of mature osteoblasts. Therefore, our results indicate that Runx2 is weakly expressed in the LepR+ population without osteoblastic commitment, and the LepR+Runx2-GFPlow stromal cells sit atop the BM stromal hierarchy.

Islet cell hyperplasia in transgenic mice overexpressing EAT/mcl-1, a bcl-2 related gene.

EAT/mcl-1 (EAT), a bcl-2 related anti-apoptotic gene, is up-regulated at the early stage of differentiation of human embryonal carcinoma cells; cells which serve as a model for early embryogenesis. We generated transgenic mice for the human EAT gene driven by the EF1 alpha promoter in order to elucidate its functional role in vivo. Histologically, these mice exhibited hyperplasia of Langerhans islet cells; pancreatic cell regions composed of both insulin- and glucagon-producing cells. Furthermore, Bax and Bag-1 -- possible heterodimeric partners for EAT in the anti-apoptotic process -- were up-regulated in islets isolated from the EAT transgenic mice. The insulin tolerance test exhibited no significant difference between the EAT transgenic mice and non-transgenic mice, indicating that islet cell hyperplasia was not due to insulin resistance. In conclusion, EAT transgenic mice exhibit hyperplasia of pancreatic beta cells. EAT may inhibit apoptosis of beta cells, allowing these cells to circumvent the process of apoptosis until the adult stage.

Penetration of drug resistance in Escherichia coli isolated from laboratory animals.

A total of 713 strains of fecal Escherichia coli (E. coli) isolated from laboratory animals in the colonies of 4 research laboratories and 4 commercial breeders in Japan in 1994 were examined in regard to resistance to 8 antibacterial agents. The incidence of resistance to sulfadimethoxine (Su), streptomycin (Sm), ampicillin, cephaloridine, tetracycline, chloramphenicol, kanamycin, and gentamicin was 99.9%, 32.5%, 6.7%, 0.7%, 7.0%, 2.6%, 6.6% and 0.7%, respectively. These results indicated that Su and Sm resistance are penetrating into normal E. coli strains isolated from laboratory animals.

Transgenic rescue of desmoglein 3 null mice with desmoglein 1 to develop a syngeneic mouse model for pemphigus vulgaris.

BACKGROUND: An active disease mouse model of pemphigus vulgaris (PV) was developed using the adoptive transfer of splenocytes from Dsg3(-/-) mice with a mixed C57BL/6J (B6) and 129/Sv genetic background into B6-Rag2(-/-) mice. Further immunological investigation is needed to resolve the genetic mismatch between host and recipient mice. The B6-Dsg3(-/-) mice did not grow old enough to provide splenocytes, probably due to severe oral erosions, with resulting inhibition of food intake. OBJECTIVE: To rescue the B6-Dsg3(-/-) mice and to produce syngeneic PV model mice. METHODS: Transgenic expression of mouse Dsg1 was attempted to compensate for the genetic loss of Dsg3 using the keratin 5 promoter. We evaluated the compensatory ability of Dsg1 in vivo by comparing Dsg1(wt/wt), Dsg1(tg/wt), and Dsg1(tg/tg) mice. We generated a PV model via the adoptive transfer of B6-Dsg1(tg/tg)Dsg3(-/-) splenocytes to B6-Rag2(-/-) mice. RESULTS: Dsg1(tg/tg) and Dsg1(tg/wt) mice expressed ectopic Dsg1 on keratinocyte cell surfaces in the lower layers of the epidermis, oral epithelium, and telogen hair follicles. Ectopic Dsg1 blocked the pathogenic effects of AK23 anti-Dsg3 mAb, and improved the body weight loss, telogen hair loss, and survival rate dose-dependently. While the B6-Dsg1(wt/wt)Dsg3(-/-) mice died by week 2, over 80% of the B6-Dsg1(tg/tg)Dsg3(-/-) mice survived at week 6. Furthermore, the syngeneic PV model mice showed the characteristic phenotype, including stable anti-Dsg3 antibody production and suprabasilar acantholysis on histology. CONCLUSION: Transgenic expression of Dsg1 rescued the severe B6-Dsg3(-/-) phenotype and provided a syngeneic mouse model of PV, which may be a valuable tool for clarifying immunological mechanisms in autoimmunity and tolerance of Dsg3.

Fra-1/AP-1 impairs inflammatory responses and chondrogenesis in fracture healing.

Inflammation inevitably follows injury of various tissues, including bone. Transgenic overexpression of Fra-1, a component of the transcription factor activator protein-1 (AP-1), in various tissues progressively and globally enhances bone formation, but little is known about the possible effects of Fra-1/AP-1 on fracture healing. We created a transverse fracture of the mouse tibial diaphysis and examined fracture healing radiologically, histologically, and immunologically. Strikingly, fracture union was delayed even though the bone formation rate in callus was higher in Fra-1 transgenic (Tg) mice. In these mice, chondrogenesis around the fracture site was impaired, resulting in accumulation of fibrous tissue, which interferes with the formation of a bony bridge across the callus. Curiously, immediately after fracture, induction of the inflammatory mediators TNF-alpha, interleukin (IL)-6, and Cox-2 was significantly suppressed in Fra-1 Tg mice followed, by the reduced expression of Sox-9 and BMP-2. Because serum prostaglandin E(2) (PGE(2)) levels were dramatically low in these mice, we administered PGE(2) to the fracture site using a slow-release carrier. The accumulation of fibrous tissue in Fra-1 Tg mice was significantly reduced by PGE(2) administration, and chondrogenesis near the fracture site was partially restored. These data suggest that the Fra-1-containing transcription factor AP-1 inhibits fracture-induced endochondral ossification and bony bridge formation presumably through suppression of inflammation-induced chondrogenesis.

SHP2-mediated signaling cascade through gp130 is essential for LIF-dependent I CaL, [Ca2+]i transient, and APD increase in cardiomyocytes.

Leukemia inhibitory factor (LIF), a cardiac hypertrophic cytokine, increases L-type Ca(2+) current (I(CaL)) via ERK-dependent and PKA-independent phosphorylation of serine 1829 in the Cav(1.2) subunit. The signaling cascade through gp130 is involved in this augmentation. However, there are two major cascades downstream of gp130, i.e. JAK/STAT3 and SHP2/ERK. In this study, we attempted to clarify which of these two cascades plays a more important role. Knock-in mouse line, in which the SHP2 signal was disrupted (gp130(F759/F759) group), and wild-type mice (WT group) were used. A whole-cell patch clamp experiment was performed, and intracellular Ca(2+) concentration ([Ca(2+)](i) transient) was monitored. The I(CaL) density and [Ca(2+)](i) transient were measured from the untreated cells and the cells treated with LIF or IL-6 and soluble IL-6 receptor (IL-6+sIL-6r). Action potential duration (APD) was also recorded from the ventricle of each mouse, with or without LIF. Both LIF and IL-6+sIL-6r increased I(CaL) density significantly in WT (+27.0%, n=16 p<0.05, and +32.2%, n=15, p<0.05, respectively), but not in gp130(F759/F759) (+9.4%, n=16, NS, and -6.1%, n=13, NS, respectively). Administration of LIF and IL-6+sIL-6r increased [Ca(2+)](i) transient significantly in WT (+18.8%, n=13, p<0.05, and +32.0%, n=21, p<0.05, respectively), but not in gp130(F759/F759) (-3.8%, n=7, NS, and -6.4%, n=10, NS, respectively). LIF prolonged APD(80) significantly in WT (10.5+/-4.3%, n=12, p<0.05), but not in gp130(F759/F759) (-2.1+/-11.2%, n=7, NS). SHP2-mediated signaling cascade is essential for the LIF and IL-6+sIL-6r-dependent increase in I(CaL), [Ca(2+)](i) transient and APD.

Three synonymous genes encode calmodulin in a reptile, the Japanese tortoise, Clemmys japonica

Three distinct calmodulin (CaM)-encoding cDNAs were isolated from a reptile, the Japanese tortoise (Clemmys japonica), based on degenerative primer PCR. Because of synonymous codon usages, the deduced amino acid (aa) sequences were exactly the same in all three genes and identical to the aa sequence of vertebrate CaM. The three cDNAs, referred to as CaM-A, -B, and -C, seemed to belong to the same type as CaMI, CaMII, and CaMIII, respectively, based on their sequence identity with those of the mammalian cDNAs and the glutamate codon biases. Northern blot analysis detected CaM-A and -B as bands corresponding to 1.8 kb, with the most abundant levels in the brain and testis, while CaM-C was detected most abundantly in the brain as bands of 1.4 and 2.0 kb. Our results indicate that, in the tortoise, CaM protein is encoded by at least three non-allelic genes, and that the æmultigene-one protein' principle of CaM synthesis is applicable to all classes of vertebrates, from fishes to mammals