Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor.

OBJECTIVE: Chondrocyte differentiation is crucial for long bone growth. Many cartilage extracellular matrix (ECM) proteins reportedly contribute to chondrocyte differentiation, indicating that mechanisms underlying chondrocyte differentiation are likely more complex than previously appreciated. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor normally abundantly produced in mesenchymal lineage cells such as adipocytes and fibroblasts, but its loss contributes to the pathogenesis of lifestyle- or aging-related diseases. However, the function of ANGPTL2 in chondrocytes, which are also differentiated from mesenchymal stem cells, remains unclear. Here, we investigate whether ANGPTL2 is expressed in or functions in chondrocytes. METHODS: First, we evaluated Angptl2 expression during chondrocyte differentiation using chondrogenic ATDC5 cells and wild-type epiphyseal cartilage of newborn mice. We next assessed ANGPTL2 function in chondrogenic differentiation and associated signaling using Angptl2 knockdown ATDC5 cells and Angptl2 knockout mice. RESULTS: ANGPTL2 is expressed in chondrocytes, particularly those located in resting and proliferative zones, and accumulates in ECM surrounding chondrocytes. Interestingly, long bone growth was retarded in Angptl2 knockout mice from neonatal to adult stages via attenuation of chondrocyte differentiation. Both in vivo and in vitro experiments show that changes in ANGPTL2 expression can also alter p38 mitogen-activated protein kinase (MAPK) activity mediated by integrin α5ß1. CONCLUSION: ANGPTL2 contributes to chondrocyte differentiation and subsequent endochondral ossification through α5ß1 integrin and p38 MAPK signaling during bone growth. Our findings provide insight into molecular mechanisms governing communication between chondrocytes and surrounding ECM components in bone growth activities.

Mechanisms inducing autonomic dysreflexia during urinary bladder distention in rats with spinal cord injury.

OBJECTIVES: This study investigated the mechanisms inducing autonomic dysreflexia due to enhanced bladder-to-vascular reflexes in rats with spinal cord injury (SCI). METHODS: SCI was produced by the transection of the Th4-5 spinal cord in female Sprague-Dawley rats. At 4 weeks after SCI, changes in blood pressure during graded increases in intravesical pressure (20-60 cm H2O) were measured in spinal-intact (SI) and SCI rats under urethane anesthesia. In five animals, effects of C-fiber desensitization induced by intravesical application of resiniferatoxin (RTX), a TRPV1 agonist, on the bladder-to-vascular reflex were also examined. Nerve growth factor (NGF) levels of mucosa and detrusor muscle layers of the bladder were measured by enzyme-linked immunosorbent assay. The expression levels of TRPV1 and TRPA1 channels were also examined in laser captured bladder afferent neurons obtained from L6 DRG, which were labeled by DiI injected into the bladder wall. RESULTS: In SI and SCI rats, systemic arterial blood pressure was increased in a pressure-dependent manner during increases in the intravesical pressure, with significantly higher blood pressure elevation at the intravesical pressure of 20 cm H2O in SCI rats vs SI rats. The arterial blood pressure responses to bladder distention were significantly reduced by RTX-induced desensitization of C-fiber bladder afferent pathways. SCI rats had higher NGF protein levels in the bladder and higher TRPV1 and TRPA1 mRNA levels in bladder afferent neurons compared with SI rats. CONCLUSIONS: The bladder-to-vascular reflex induced by TRPV1-expressing C-fiber afferents during bladder distention is enhanced after SCI in association with increased expression of NGF in the bladder and TRP channels in bladder afferent neurons.

Fgf10 is essential for limb and lung formation.

The interactions between fibroblast growth factors (FGF) and their receptors have important roles in mediating mesenchymal-epithelial cell interactions during embryogenesis. In particular, Fgf10 is predicted to function as a regulator of brain, lung and limb development on the basis of its spatiotemporal expression pattern in the developing embryo. To define the role of Fgf10, we generated Fgf10-deficient mice. Fgf10-/- mice died at birth due to the lack of lung development. Trachea was formed, but subsequent pulmonary branching morphogenesis was disrupted. In addition, mutant mice had complete truncation of the fore- and hindlimbs. In Fgf10-/- embryos, limb bud formation was initiated but outgrowth of the limb buds did not occur; however, formation of the clavicles was not affected. Analysis of the expression of marker genes in the mutant limb buds indicated that the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) did not form. Thus, we show here that Fgf10 serves as an essential regulator of lung and limb formation.

Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning.

1 alpha,25-Dihydroxyvitamin D3[1 alpha,25(OH)2D3], an active form of vitamin D, has roles in many biological phenomena such as calcium homeostasis and bone formation, which are thought to be mediated by the 1 alpha,25(OH)2D3 receptor (VDR), a member of the nuclear hormone receptor superfamily. However, the molecular basis for the actions of 1 alpha,25(OH)2D3 in bone formation, its role during development and VDR genetic polymorphisms for predicting bone mineral density are uncertain. To investigate the functional role of VDR, we generated mice deficient in VDR by gene targeting. We report here that in VDR null mutant mice, no defects in development and growth were observed before weaning, irrespective of reduced expression of vitamin D target genes. After weaning, however, mutants failed to thrive, with appearance of alopoecia, hypocalcaemia and infertility, and bone formation was severely impaired as a typical feature of vitamin D-dependent rickets type II (refs 8, 9). Unlike humans with this disease, most of the null mutant mice died within 15 weeks after birth, and uterine hypoplasia with impaired folliculogenesis was found in female reproductive organs. These defects, such as alopoecia and uterine hypoplasia, were not observed in vitamin D-deficient animals. The findings establish a critical role for VDR in growth, bone formation and female reproduction in the post-weaning stage.

Endothelin: a novel peptide in the posterior pituitary system.

Endothelin (ET), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, is present in the porcine spinal cord and may act as a neuropeptide. Endothelin-like immunoreactivity has now been demonstrated by immunohistochemistry in the paraventricular and supraoptic nuclear neurons and their terminals in the posterior pituitary of the pig and the rat. The presence of ET in the porcine hypothalamus was confirmed by reversed-phase high-pressure liquid chromatography and radioimmunoassay. Moreover, in situ hybridization demonstrated ET messenger RNA in porcine paraventricular nuclear neurons. Endothelin-like immunoreactive products in the posterior pituitary of the rat were depleted by water deprivation, suggesting a release of ET under physiological conditions. These findings indicate that ET is synthesized in the posterior pituitary system and may be involved in neurosecretory functions.

T-type calcium channel blockade as a therapeutic strategy against renal injury in rats with subtotal nephrectomy.

T-type calcium channel blockers have been previously shown to protect glomeruli from hypertension by regulating renal arteriolar tone. To examine whether blockade of these channels has a role in protection against tubulointerstitial damage, we used a stereo-selective T-type calcium channel blocker R(-)-efonidipine and studied its effect on the progression of this type of renal injury in spontaneously hypertensive rats that had undergone subtotal nephrectomy. Treatment with racemic efonidipine for 7 weeks significantly reduced systolic blood pressure and proteinuria. The R(-)-enantiomer, however, had no effect on blood pressure but significantly reduced proteinuria compared to vehicle-treated rats. Both agents blunted the increase in tubulointerstitial fibrosis, renal expression of alpha-smooth muscle actin and vimentin along with transforming growth factor-beta (TGF-beta)-induced renal Rho-kinase activity seen in the control group. Subtotal nephrectomy enhanced renal T-type calcium channel alpha1G subunit expression mimicked in angiotensin II-stimulated mesangial cells or TGF-beta-stimulated proximal tubular cells. Our study shows that T-type calcium channel blockade has renal protective actions that depend not only on hemodynamic effects but also pertain to Rho-kinase activity, tubulointerstitial fibrosis, and epithelial-mesenchymal transitions.

Intron retention generates a novel isoform of the murine vitamin D receptor that acts in a dominant negative way on the vitamin D signaling pathway.

We identified and characterized a novel rat vitamin D receptor isoform (rVDR1), which retains intron 8 of the canonical VDR (rVDR0) during alternative splicing. In this isoform protein directed by the stop codon in this newly identified exon, a part of the ligand binding domain (86 amino acids) is truncated at the C-terminal end but contains 19 extra amino acids. The rVDR1 transcript was expressed at a level 1/15 to 1/20 of that of rVDR0 in the kidney and intestine in adult rats but not in embryos. The recombinant rVDR1 protein showed no ligand binding activity. Homo- and heterodimers of the recombinant rVDR0 and rVDR1 proteins bound to a consensus vitamin D response element (VDRE) but not to consensus response elements for thyroid hormone and retinoic acid. However, unlike rVDR0, rVDR1 did not form a heterodimeric complex with RXR on the VDRE. A transient expression assay showed that this isoform acted as a dominant negative receptor against rVDR0 transactivation. Interestingly, the dominant negative activities of rVDR1 differed among VDREs. Thus, the present study indicates that this new VDR isoform negatively modulates the vitamin D signaling pathway, through a particular set of target genes.

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. II.

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5-20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0 degrees C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at -4 degrees C. Thus, we conclude that the phosphodiesterase in activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.

Two forms of intermediates of frog rhodopsin in rod outer segments.

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at -190 degrees C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at -190 degrees C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho1-rhodopsin and batho2-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at -190 degrees C. We infer that a rhodopsin molecule in the excited state relaxes to either batho1-rhodopsin or batho2-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.

Circular dichroism of squid rhodopsin and its intermediates.

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates was measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the alpha- and beta-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at alpha-band and 348 nm at beta-band) as well as at room temperature (CD maxima: 474 nm at alpha-band and 347 nm at beta-band). The rotational strength of the alpha-band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at alpha-band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may de derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, The CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.

Circular dichroism of cattle rhodopsin and bathorhodopsin at liquid nitrogen temperatures.

The photoevent in vision has been considered to be the conversion of rhodopsin to bathorhodopsin, which is caused by photoisomerization of the chromphoric retinal. Recently some objections were raised to this hypothesis. The reliability of the hypothesis was verified by measurement of circular dichroism of bathorhodopsin. The measurement of circular dichroism of rhodopsin extract (containing 66% or 75% of glycerol) at liquid nitrogen temperatures (-195 degrees C) by a conventional spectropolarimeter induced an extraordinary large signal, owing to linear dichroism originated from conversion of rhodopsin to bathorhodopsin by the measuring light. The similar linear dichrolism can be induced by irradiation of rhodopsin extract at -195 degrees C with polarized light or natural light. At photosteady state the linear dichroism disappeared. Circular dichroism spectrum of cattle rhodopsin displayed two positive peaks ([theta]max = 80 800 degrees at 335 nm, and [theta]max = 42 600 degrees at 500 nm) at -195 degrees C, whereas, bathorhodopsin displayed a positive peak ([theta]max = 43 100 degrees at 334 nm) and a negative peak ([theta]max = 163 000 degrees at 540 nm). The change of the positive sign to negative one at alpha-band of circular dichroism spectrum supports the hypothesis that the conversion of rhodopsin is due to rotation of the chromophoric retinal about C-11--12 double bond ('photoisomerization model').

Accessibility of the iodopsin chromophore.

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from iodopsin in the dark. These facts suggest that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.

Formation of hypsorhodopsin at room temperature by picosecond green pulse.

Excitation of squid rhodopsin with a single laser pulse (532 nm, 25 ps) at 18 degrees C yielded photorhodopsin, a precursor of bathorhodopsin. In the linear region, no relation between amount of photorhodopsin and excitation-energy hypsorhodopsin was detected, while in a photon saturation region this was observed. The time constant of hypsorhodopsin to bathorhodopsin decay was about 125 ps. Dependencies of formation of photorhodopsin and hypsorhodopsin on the excitation energy suggest that hypsorhodopsins of squid and octopus are formed by a two-photon reaction. No cattle hypsorhodopsin was detected in our experimental conditions.

Formation of hyposorhodopsin in frog retina.

Hypsorhodopsin was formed in frog retina by irradiation at liquid helium temperature and converted into bathorhodopsin above about 29K.

Activation of phosphodiesterase in frog rod outer segment by rhodopsin analogues.

Activation of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35) in frog rod outer segment membrane by rhodopsin analogues has been investigated. A rhodopsin analogue modified at the Schiff-base linkage (N-retinyl-opsin) or the beta-ionone ring (3-dehydro-rhodopsin) in the retinylidene chromophore of rhodopsin has some ability in activation of the enzyme. In consideration of our previous observation that opsin including a retinal-oxime can activate the enzyme, it seems likely that the Schiff-base linkage is not always necessary for the phosphodiesterase activation. On the other hand, a change in the length of the side chain of retinal (complex of opsin and beta-ionone, beta-ionylideneacetaldehyde or retinylideneacetaldehyde) or dissection of the conjugate double-bond system of the side chain (retro-gamma-rhodopsin) remarkably reduces the activation ability. However, 5,8-epoxy-rhodopsin having a similar dissected conjugate double-bond system induces some enzyme activation because of its rigid conformation around C7-C8-C9 single bonds. Consequently, it is suggested that the necessary portion of rhodopsin chromophore for the activation of the enzyme is the rigid conjugate double-bond system between the beta-ionone ring and the Schiff-base linkage in its all-trans form.

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis I.

Guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concanavalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the crude rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphodiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.

Photochemical reaction of 9-cis-retro-gamma-rhodopsin at low temperatures.

9-cis-Retro-gamma-rhodopsin (lambda max = 420 nm) was prepared from 9-cis-retro-gamma-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-gamma-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-gamma-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of batho-rhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.

Photochemical reaction of 7,8-dihydrorhodopsin at low temperatures.

The photoreaction of 9-cis-7,8-dihydrorhodopsin was examined at liquid nitrogen temperatures (-180 degrees C) in order to elucidate the photochemical events in visual pigments. This rhodopsin analog was prepared by incubating 9-cis-7,8-dihydroretinal with bovine opsin in the dark. 9-cis-7,8-Dihydrorhodopsin (lambda max = 427 nm) was cooled to -180 degrees C, and then irradiated at -180 degrees C with a 390 nm light, resulting in formation of its bathochromic product (lambda max = 465 nm). This result indicates that the presence of four double-bonds adjacent to the Schiff base nitrogen is sufficient to allow formation of a bathochromic product. Thus, the mechanism of formation of bathorhodopsin (in bovine rhodopsin system) may be considered as some change of the interaction between the conjugated double-bond system from C-9 to the Schiff base nitrogen and its surrounding charges in opsin, caused by rotation of 11-12 double-bond.