In vitro proteoglycan synthesis in response to extracts of demineralized bone.

Two extracts of bovine bone, bone morphogenetic protein (BMP) supplied by the UCLA Bone Research Laboratory, and osteogenic factor extract (OFE) supplied by the industrial group Celtrix Pharmaceuticals, were tested for the ability to transform embryonic skeletal muscle into cartilage. Skeletal muscle was placed into organ cultures on substrata of Type I collagen and fed with concentrations of the extracts that their originators reported to be effective; however, only BMP was capable of eliciting the morphologic differentiation of cartilage. In contrast, both extracts supported patterns of glycosaminoglycan synthesis that mimicked the biochemical differentiation of cartilage-type extracellular matrix. Bone morphogenetic protein differed from OFE in its ability to elicit high levels of hyaluronic acid synthesis, although BMP and OFE upregulated synthesis of hyaluronic acid that was of sufficient chain length to support proteoglycan aggregate formation. Proteoglycan extracts of the cell layer and medium demonstrated that most of the proteoglycan synthesized in response to BMP was an aggrecan-like material, which was lost to the medium. That which synthesized in response to OFE was a proteoglycan with short glycosaminoglycan chains that had only a limited ability to aggregate. These results demonstrate that BMP is effective in promoting chondrogenesis by virtue of its ability to promote the synthesis of hyaluronic acid, and aggrecan, but suggests that other accessory matrix components must also be synthesized to anchor aggrecan in the cell layer. The ability to stimulate the synthesis of these other components may be lost on purification of BMP. Consequently, BMP may initiate several activities that collectively upregulate chondrogenesis and the production of cartilage extracellular matrix.

Altered patterns of proteoglycan deposition during maturation of the fetal mouse lung.

Previous studies have shown that beta-xyloside inhibits maturation of the fetal mouse lung (Smith et al., Dev. Biol. 138, 42-52, 1990). Insofar as this drug inhibits proteoglycan deposition, the present studies were undertaken to examine the chemical composition and tissue distribution of proteoglycans in order to determine, more precisely, their role during lung morphogenesis. Autoradiography of labeled 16- and 19-day embryonic lungs demonstrated greater incorporation over the mesenchyme. Treatment with beta-xyloside did not alter the autoradiographic appearance; however, beta-xyloside treatment followed by nitrous acid digestion, eliminated most silver grains. Isolation of proteoglycans from extracellular, membrane and intracellular pools over the 16- to 19-day interval demonstrated redistribution of heparan sulfate proteoglycan from an intracellular to a membrane location, while chondroitin sulfate proteoglycan redistributed from intracellular to extracellular. Only the synthesis of chondroitin sulfate proteoglycan was inhibited by beta-xyloside. On the basis of these results we suggest that a chondroitin sulfate proteoglycan is required for lung maturation and that inhibition of its synthesis results in inhibition of septa formation and subsequent failure of morphogenesis and differentiation.

Effects of beta-D-xyloside on differentiation of the respiratory epithelium in the fetal mouse lung.

Differentiation of respiratory endings in the fetal lung appears to be controlled by its surrounding mesodermal capsule. The capsule may exert its influence by controlling the composition of the epithelial basal lamina or of the extended extracellular matrix that is deposited during the period when alveolar sacs are formed. As a first step in testing this hypothesis, the effects of the drug, rho-nitrophenyl-beta-D- xylopyranoside (beta-xyloside), an inhibitor of proteoglycan synthesis, and its inactive alpha anomer (alpha-xyloside) were examined. Lung primordia from mice at 16 days of gestation were tested for inhibition of morphological and functional differentiation as a result of drug treatment. Pseudoglandular lung epithelium did not form respiratory endings, contained fewer specialized cells, and accumulated little additional surfactant when treated with beta-xyloside but developed normally when treated with alpha-xyloside or grown in control medium. The results are interpreted to suggest that deposition of an extracellular matrix rich in proteoglycan is required to support maturation of the respiratory epithelium.

Hyaluronates in developing skeletal tissues.

Hyaluronic acid (HA) is present in the extracellular matrix (ECM) as early as the time of gastrulation. At these early stages, HA is thought to organize the ECM into a hydrated, open lattice and thereby support cell movements. At later stages, when specialized tissues appear, hyaluronidase activity increases. A correlation between elevated hyaluronidase activity and deposition of cartilage-type ECM is now well established. Differentiation of cartilage may be accompanied by changes in molecular forms of HA; however, the synthesis of HA is not understood well enough to permit firm generalizations to be drawn. There is also a lack of evidence regarding specificity of the hyaluronidase that appears at the onset of chondrogenesis. Thus, while HA is a ubiquitous ECM component and its appearance has been well studied during embryogenesis, there remain large gaps in the present knowledge regarding the means by which HA interacts with embryonic cells, tissues, and other ECM components.

Posttranscriptional control of embryonic rat skeletal muscle protein synthesis. Control at the level of translation by endogenous RNA.

The onset of muscle cell differentiation is associated with increased transcription of muscle-specific mRNA. Studies from this laboratory using 19-d embryonic rat skeletal muscle, suggest that additional, posttranscriptional controls regulate maturation of muscle tissue via a quantitative effect upon translation, and that the regulatory component may reside within the poly A- RNA pool (Nathanson, M.A., E.W. Bush, and C. Vanderburg. 1986. J. Biol. Chem. 261:1477-1486). To further characterize muscle cell translational control, embryonic and adult total RNA were separated into oligo(dT)cellulose-bound (poly A+) and -unbound (poly A-) pools. Unbound material was subjected to agarose gel electrophoresis to resolve constituents of varying molecular size and mechanically cut into five fractions. Material of each fraction was electroeluted and recovered by precipitation. Equivalent loads of total RNA from 19-20-d embryonic rat skeletal muscle exhibited a 40% translational inhibition in comparison to its adult counterpart. Inhibition was not due to decreased message abundance because embryonic, as well as adult muscle, contained equivalent proportions of poly A+ mRNA. An inhibition assay, based upon the translatability of adult RNA and its inhibition by embryonic poly A- RNA, confirmed that inhibition was associated with a 160-2,000-nt poly A- fraction. Studies on the chemical composition of this fraction confirmed its RNA composition, the absence of ribonucleoprotein, and that its activity was absent from similarly fractionated adult RNA. Rescue of inhibition could be accomplished by addition of extra lysate or mRNA; however, smaller proportions of lysate were required, suggesting a strong interaction of inhibitor and components of the translational apparatus. Additional studies demonstrated that the inhibitor acted at the level of initiation, in a dose-dependent fashion. The present studies confirm the existence of translational control in skeletal muscle and suggest that it operates at the embryonic to adult transition. A model of muscle cell differentiation, based upon transcriptional control at the myoblast level, followed by translational regulation at the level of the postmitotic myoblast and/or myotube, is proposed.

Human dentin matrix induces cartilage formation in vitro by mesenchymal cells derived from embryonic muscle.

Dentin matrix was assayed for its potential to elicit chondrogenesis of mesenchymal cells in vitro. The substratum was prepared by demineralization of human tooth root dentin, while embryonic thigh muscle was used as a source of mesenchymal cells. Formation of chondrocytes from mesenchymal cells occurred in the presence of dentin matrix, and in the same sequence as previously shown with substrata of demineralized bone.

Transcriptional-translational regulation of muscle-specific protein synthesis and its relationship to chondrogenic stimuli.

Demineralized bone (bone matrix) has a well-characterized ability to evoke the re-differentiation of cells derived from skeletal muscle into chondrocytes. Recent investigations in this laboratory have shown that muscle-specific (alpha) actin synthesis continues throughout redifferentiation. Conversely, expression of the cartilage phenotype is associated with repression of muscle-specific enzyme synthesis. The present experiments were undertaken to determine the mode of genomic regulation responsible for control of these muscle-specific syntheses. As part of these experiments, we investigated the ability of embryonic and adult RNA to direct translation in vitro. The results indicate that unfractionated (total) RNA is capable of directing the efficient synthesis of actin, but not myosin heavy or light chains. Decreased abundance of polyadenylated mRNA cannot account for lack of myosin synthesis. Polyadenylated mRNA, however, directed synthesis of actin and myosin with an efficiency greater than that of total RNA. This data suggested that embryonic total RNA was subject to translational control. Dot blot hybridization against cDNA probes for alpha-actin, myosin heavy chain, and fast light chains demonstrated that myogenic cells were subject to a pattern of mixed transcriptional and translational control. It is hypothesized that full expression of the muscle phenotype involves sequential release of transcriptional, and subsequently, the translational controls. We have also observed that cultures of skeletal muscle on bone matrix contain mRNA for muscle-specific proteins, even through the period normally characterized by chondrogenesis. In the absence of concurrent enzyme protein synthesis, it appears that one action of bone matrix is to continue genomic controls that in the source skeletal muscle maintain the genome in an embryonic (translationally repressed) state.

Bone matrix-directed chondrogenesis of muscle in vitro.

Bone matrix is the largely collagenous residue of demineralized bone. Experimental data demonstrate that a substance, which is acid-stable during demineralization, occurs as a part of bone matrix, and that it is capable of stimulating the redifferentiation of skeletal muscle into cartilage. Reproducibility of redifferentiation is high and all cells derived from embryonic mesoderm appear competent to yield cartilage. This effect is highly significant to the developmental biology of musculoskeletal tissues, as muscle and cartilage arise from a similar embryonic origin. With regard to the embryonic limb as a model system, it appears that both muscle and cartilage progenitor cells do not have rigidly-defined developmental programs, and that this is a result of their origin from a common pool of embryonic mesoderm. This pool originates as embryonic mesenchyme long before any evidence of limb development can be detected. It is proposed that the active component of bone matrix, termed "bone morphogenetic protein (BMP)," acts upon a tissue whose developmental program is not stabilized, or has been experimentally destabilized (by injury), to augment and sustain syntheses of cartilage extracellular matrix. The use of bone matrix, and active substances derived from it, suggests that differentiation is not irreversible. Hard tissue growth and repair may occur via recruitment of competent responding cells from a variety of nonchondrogenic sources, provided that the extracellular milieu (i.e., presence of BMP) is supportive.

In vitro transformation of mesenchymal cells derived from embryonic muscle into cartilage in response to extracellular matrix components of bone.

Subcutaneous implantation of demineralized diaphyseal bone matrix into rats induces cartilage and bone formation in vivo. When minced skeletal muscle is cultured on hemicylinders of demineralized bone in vitro, mesenchymal cells are transformed into chondrocytes. In the present investigation, the potential of extracellular matrix components of bone to trigger cartilage differentiation in vitro was examined. Extraction of bone hemicylinders with 6 M guanidine X HCl resulted in the absence of chondrogenesis in vitro and endochondral bone formation in vivo. Biologically inactive hemicylinders of bone were then reconstituted with the guanidine extract and also with partially purified components extracted from bone matrix and bioassayed. Reconstitution completely restored the ability to elicit chondrogenesis in vitro and endochondral bone differentiation in vivo. Reconstitution of the whole guanidine extract on Millipore filters coated with gels of tendon collagen (type I) and subsequent culture with minced skeletal muscle also resulted in cartilage induction in vitro. These observations show that the extracellular matrix of bone is a repository of factors that govern local cartilage and bone differentiation.

Proteoglycan synthesis by skeletal muscle undergoing bone matrix-directed transformation into cartilage in vitro.

Myoblasts and fibroblasts of embryonic skeletal muscle reproducibly form chondrocytes when cultured on demineralized bone in vitro. The transformation occurs in 3 morphologically defined phases, with disappearance of the myoblast phenotype preceding the appearance of fibroblast-like cells and finally chondrocytes. Proteoglycan synthesis in these cultures was investigated by labeling at prechondrogenic (5 days) and postchondrogenic (6-12 days) stages with (35S)sulfate and [6-3H]glucosamine. Labeled material elutes from associative Sepharose CL-2B columns as two major included peaks, which correspond to proteoglycan monomer and a material of lower molecular size. Control cultures, cultured upon gels of type I collagen, fail to synthesize monomer-like material and contain solely a material of lower molecular size. Demineralized bone-derived monomer was rechromatographed under dissociative conditions in an attempt to detect the presence of small aggregates. Again, only a single peak of sulfate and glucosamine-labeled material appears. The data further show that the monomer resembles that of embryonic cartilage in glycosaminoglycan chain size (Mr = 8.6-12.2 X 10(3] and composition (mainly chondroitin 4-sulfate). Aggregated monomer forms a shoulder of the monomeric peak and comprises only 5% of the sulfated material. Fifteen to thirty-four per cent of the monomer elutes as aggregate after addition of rooster comb hyaluronic acid (HA). Failure to aggregate appears to be related to endogenous synthesis of short chain HA. Synthesis of long chain HA may constitute a rate-limiting step in chondrogenesis. Material of lower molecular size, from cultures grown on demineralized bone, bind to exogenous HA, whereas the elution pattern of sulfated material from control cultures remains essentially unchanged. These latter data suggest that proteoglycans of low hydrodynamic size may participate in the early formation of proteoglycan aggregate.

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix. III. Environmental regulation of glycosaminoglycan and proteoglycan synthesis.

The ability of numerous nutritional and topographic factors to influence differentiation of embryonic mesenchyme has given rise to several theories which attempt to explain the development of muscle and cartilage from these similar-appearing cells. Some theories are challenged by the observation that a substratum of demineralized bone is capable of supporting the transformation of skeletal muscle into cartilage in vitro and that the potential to form cartilage still resides within cloned myoblasts and fibroblasts of skeletal muscle. In the present study, culture media CMRL-1066, minimal essential medium (MEM), and F-12 provide varied nutritional environments and are tested for their ability to support the morphological and biochemical transformation of skeletal muscle into cartilage. Morphologically, CMRL-1066 reproducibly supports hyaline cartilage formation, whereas MEM does so in only one out of three explants onto demineralized bone, and F-12 is incapable of supporting formation of a hyaline matrix. Biochemically, each medium is sufficient to elicit synthesis of cartilage-like patterns of sulfated glycosaminoglycans and proteoglycan monomer. Synthesis of hyaluronic acid (HA) initially increases in explants grown in CMRL-1066, but decreases prior to chondrogenesis. MEM elicits a similar increase in HA synthesis, but the subsequent decrease is not as rapid. In F-12, synthesis remains depressed throughout the experiment. The data show that increases in HA synthesis occur concurrent with the appearance of fibroblast-like cells, which normally precede chondroblasts. Decreases in HA synthesis correlate well with the onset of chondrogenesis. Explants grown in CMRL-1066 reproducibly from cartilage and synthesize the greatest amounts of proteoglycan aggregate. Those grown in MEM form cartilage infrequently, synthesize reduced amounts of proteoglycan aggregate-like material, and contain greater amounts of HA, of low molecular weight. The data demonstrate that chondrogenesis can be subtly regulated by environmental factors, and such factors regulate both the morphological and biochemical expression of the phenotype through HA synthesis.

Morphological and biochemical transformation of skeletal muscle into cartilage.

In summary, these studies show that the potential to form cartilage is not restricted to a defined population of limb tissues, but resides within limb mesenchyme as one of its several potencies. A theoretically novel aspect of these studies is the relative ease with which the synthesis of sulfated GAG and proteoglycan monomer may be evoked. But, what appears to control the appearance of a fully differentiated cartilage matrix is a shift from the synthesis of low molecular weight HA to species of greater molecular weight.

Cultivation of mammalian pineal cells: retention of organization and function in tissue culture.

By means of a newly developed method of cultivating pineal tissue in vitro, the types of cells which comprise rat pineal glands have been identified. Previous in vitro studies have involved short-term culture more suitably called "organ culture" and provide no means of assessing the contribution of a putative "pineal" cell versus any other cell type found in the cultures. Short-term outgrowths of minced rat pineal glands provided a reproducible and easily dissociated source of pineal-derived cells. In monolayer culture these cells continued to have pineal enzyme activities which were sensitive to pineal-activating substances, and the cells aggregated to mimic the lobular organization of intact glands. Two types of aggregates were found, each composed of a single morphological cell type. In addition to the transient appearance of skeletal muscle straps, connective tissue and neural/glial tissue was consistently found. The cell types are discussed in relation to their in vivo counterparts.