High-dose retinoic acid modulates rat calvarial osteoblast biology

Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis.     

Hindbrain patterning involves graded responses to retinoic acid signalling

Several recent studies have shown that retinoic acid signalling is required for correct patterning of the hindbrain. However, the data from these studies are disparate and the precise role of retinoic acid signalling in patterning the anteroposterior axis of the neural tube remains uncertain. To help clarify this issue, we have cultured a staged series of chick embryos in the presence of an antagonist to the all three retinoic acid receptors. Our data indicate that retinoic acid is the transforming signal involved in the expansion of posterior hindbrain structures. We find that the hindbrain region of the neural tube down to the level of the sixth somite acquires the identity of rhombomere 4 when retinoic acid signalling is blocked. Specification of future rhombomere boundaries has a retinoic acid dependency between stage 5 and stage 10(+) that is lost progressively in an anterior-to-posterior sequence. Furthermore, the application of various concentrations of antagonist shows that successively more posterior rhombomere boundaries require progressively higher concentration of endogenous retinoic acid for their correct positioning, a result that strengthens the hypothesis that a complex retinoid gradient acts to pattern the posterior hindbrain. Our dissection of early retinoic acid functions allows us to re-interpret the wide disparity of hindbrain phenotypes previously observed in various models of retinoic acid deficiency.     

Stage-dependent responses of the developing lung to retinoic acid signaling

Morphological analysis of vitamin A-deficient rat fetuses and of retinoic acid receptor (RAR and RXR) mutant mice have demonstrated that retinoic acid (RA) is essential for lung development. To gainfurther insight into RA signaling pathways during primary lung budformation and lung branching, we have investigated the effects of RA and of a pan-RAR antagonist in cultures of whole embryos and lung explants. Treatment of E8.0 embryos with the pan-RAR antagonist inhibits the formation of the primitive respiratory system. On the other hand, treatment of E11.75 and E12.5 lung explants with RA inhibits branching morphogenesis, whereas treatment with the pan-RAR antagonist at the same developmental stages stimulates formation of distal buds. The inhibitory effect of RA on branching is strongly decreased in RARbeta null lungs, while enhancement of budding by the pan-RAR antagonist is not affected by an RARgamma null mutation. Additionally, cellular retinol binding protein one (CRBPI) null lungs are more sensitive than wild type lungs to the pan-RAR antagonist-induced stimulation of branching. These data indicate that retinoid signaling is indispensable for the formation of primary lung buds and the oesophagotracheal septum from the primitive foregut. They also suggest that at the pseudoglandular stage, RA signaling through RARbeta, but not RARgamma, inhibits distal bud formation thereby promoting the formation of conducting airways. Moreover, the level of CRBPI in the pseudoglandular lung appears to participate in the control of branching morphogenesis.     

Fatty acid synthase modulates homeostatic responses to myocardial stress

Fatty acid synthase (FAS) promotes energy storage through de novo lipogenesis and participates in signaling by the nuclear receptor PPARα in noncardiac tissues. To determine if de novo lipogenesis is relevant to cardiac physiology, we generated and characterized FAS knockout in the myocardium (FASKard) mice. FASKard mice develop normally, manifest normal resting heart function, and have normal cardiac PPARα signaling as well as fatty acid oxidation. However, they decompensate with stress. Most die within 1 h of transverse aortic constriction, probably due to arrhythmia. Voltage clamp measurements of FASKard cardiomyocytes show hyperactivation of L-type calcium channel current that could not be reversed with palmitate supplementation. Of the classic regulators of this current, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) but not protein kinase A signaling is activated in FASKard hearts, and knockdown of FAS in cultured cells activates CaMKII. In addition to being intolerant of the stress of acute pressure, FASKard hearts were also intolerant of the stress of aging, reflected as persistent CaMKII hyperactivation, progression to dilatation, and premature death by ～1 year of age. CaMKII signaling appears to be pathogenic in FASKard hearts because inhibition of its signaling in vivo rescues mice from early mortality after transverse aortic constriction. FAS was also increased in two mechanistically distinct mouse models of heart failure and in the hearts of humans with end stage cardiomyopathy. These data implicate a novel relationship between FAS and calcium signaling in the heart and suggest that FAS induction in stressed myocardium represents a compensatory response to protect cardiomyocytes from pathological calcium flux.     

The cell substratum modulates skeletal muscle differentiation

During chick embryogenesis, massive alterations occur in the migrating cell's substratum, or extracellular matrix. The possibility that some of the components of this milieu play a regulatory role in cell differentiation was explored in a cell-culture system derived from embryonic chick skeletal muscle tissue. In particular, the effects of collagen and the glycosaminoglycans were studied. Collagen is required for muscle cell attachment and spreading onto plastic and glass tissue-culture dishes. A major constituent of the early embryonic extracellular space, hyaluronate (HA), while having no significant effect on collagen-stimulated cell attachment and spreading, was found to inhibit myogenesis. The muscle-specific M subunit of creatine kinase was preferentially inhibited. Control experiments indicated that the inhibition was specifically caused by HA and not by other glycosaminoglycans. A general metabolic inhibition of the cultures was not observed. Muscle cells could bind to HA-coated beads at all stages of differentiation but were inhibited only when HA was added within the first 24 h of culture. Endogenous GAG in the culture is normally degraded during the first 24 h after plating as well; this may parallel the massive degradation of HA that occurs in the early embryo in vivo. These findings suggest a regulatory role for HA in modulating skeletal muscle differentiation, with degradation of an inhibitory component of the cell substratum a requirement for myogenesis.     

Cell attachment to a substratum and cell surface proteases.

The polytripeptide (Tyr-Ala-Glu)_n, n～-175, has been reported to undergo an α-helix-disordered chain transition in aqueous medium (Ramachandran, J., et al. (1971) Biopolymers, 10, 1829–1851). We find from circular dichroism and infrared spectroscopy that, upon transferring (Tyr-Ala-Glu)₉ from aqueous buffer at neutral pH to dioxane-containing media at acidic pH, and in certain other circumstances, a transition from the disordered state to the antiparallel β structure occurs. Molecular weight studies and the independence of the transition from concentration suggest that the β structure is intramolecular. (Tyr-Ala-Glu)₄ shows no evidence for the occurrence of any conformational change under similar conditions.     

The cryptic responses of hatchery-reared sole to a natural sand substratum

In laboratory experiments, both reared and wild sole Solea solea selected a sand substratum in preference to a hard substratum. Reared sole with no previous experience of sand buried quickly when placed on sand. Light motivated burial, and the motivation of reared sole to bury was as strong as that of wild sole. The burial efficiency (the proportion of the ocular side covered by sand after a single burial attempt) of reared sole was lower than that of wild sole, but increased to that of wild sole after a period of 12 days maintenance on sand. Motivation to bury is therefore innate, but efficiency is affected by experience. The reactive distance of reared sole to a standardized predation threat was the same as that of wild sole and was shorter when buried (6 cm) than when not buried (15 cm). Burial therefore affected the response to a predation threat, indicating that burial is a cryptic behaviour. Munsell colour charts were used to determine the time required for reared sole to match the skin colour 'tone' of wild sole after placement on sand. Adaptation of colour value (lightness) took 4–7 days, but adaptation of chroma (intensity of colour) and hue took 33 and 69 days respectively. It is therefore recommended that flatfishes reared for stock enhancement exercises are conditioned to sand prior to release due to the relatively long time required for crypsis to improve through colour adaptation and burying.     

Behavioural responses of benthic and pelagic Arctic charr to substratum heterogeneity

Responses to environmental heterogeneity were studied in laboratory-reared offspring of two morphs of Arctic charr Salvelinus alpinus from Loch Rannoch, Scotland, one occupying the pelagic zone and feeding predominantly on zooplankton and the other being benthic in habit and feeding mainly on macroinvertebrates. When housed in groups in tanks with a black-and white striped base, benthic charr demonstrated a clear preference for dark areas, whereas pelagic fish positioned themselves at random with respect to substratum colour. In general, pelagic charr were much less aggressive than benthic charr. In pelagic fish, neither spacing nor aggression was affected by the visual heterogeneity of the substrate. In contrast, benthic charr swam closer together and fought more when housed over a uniform as opposed to a non-uniform substratum. The results are discussed in the context of habitat-specific visual requirements and of an interaction between visual complexity and territoriality previously described for Atlantic salmon Salmo salar .     

Zoledronic acid modulates antitumoral responses of prostate cancer-tumor associated macrophages

Macrophages are considered a key component of the immunosuppressive environment present in solid tumors, where they support tumor growth through the production of pro-angiogenic factors and active suppression of effector immune responses. Zoledronic acid (ZA), an aminobisphosphonate clinically approved for treatment of symptomatic skeletal events, has recently been shown to have immunomodulatory properties that can be exploited in cancer immunotherapy. Here, we utilize an in vitro model of prostate cancer cell-macrophage interaction to dissect the effect of ZA, on the function of prostate cancer tumor-associated macrophages (PC-TAM). We show that prostate cancer cells recruit macrophages, which in turn express a variety of proangiogenic and immunosuppressive mediators. ZA selectively suppressed the expression of MMP-9 by PC-TAM, whereas the expression of other mediators was not limited. PC-TAM treated with ZA, on the other hand, could effectively drive the proliferation of activated Tgammadelta lymphocytes, which lysed bisphosphonate-pulsed prostate cancer cells. Moreover, ZA boosted the production of type-1 cytokines by PC-TAM in response to immunomodulators such as IL-12 and polyI:C, which are known to polarize macrophages towards an anti-tumoral M1 phenotype. Overall, we provide evidence that ZA shifts the balance of PC-TAM from a tumor promoting to a tumor-eliminating phenotype and also suggest a potential use of this pharmacological agent as an immunotherapeutic adjuvant.     

Stable,position-related responses to retinoic acid by chick limb-bud mesenchymal cells in serum-free cultures

Summary Retinoic acid (RA) has dramatic effects on limb-skeletal patterning in vivo and may well play a pivotal role in normal limb morphogenesis. RA’s effects on the expression of pattern-related genes in the developing limb are probably mediated by cytoplasmic RA-binding proteins and nuclear RA-receptors. Little is known, however, about how RA modifies specific cellular behaviors required for skeletal morphogenesis. Earlier studies supported a role for regional differences in RA concentration in generating the region-specific cell behaviors that lead to pattern formation. The present study explores the possibility that position-related, cell-autonomous differences in the way limb mesenchymal cells respond to RA might have a role in generating pattern-related cell behavior. Mesenchymal cells from different proximodistal regions of stage 21–22 and 23–24 chick wing-buds were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high-density microtiter cultures. The effects of RA on chondrogenesis in these cultures clearly differed depending on the limb region from which the cells were isolated. Regional differences in RA’s effects on growth over 4 days in these cultures were less striking. The region-dependent responses of these cells to RA proved relatively stable in culture despite ongoing cytodifferentiation. This serum-free culture model will be useful in exploring the mechanisms underlying the region-dependent responsiveness of these cells to RA.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Endoglin modulates cellular responses to TGF-beta 1

Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L- or S- endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S- endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF- beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-beta 1 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L- endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.     

Pre-existing immunity modulates responses to mRNA boosters

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