I. Introduction A FUNCTIONAL relationship between cell growth and the initiation and progression of events associated with differentiation has been a fundamental question challenging developmental biologists for more than a century. In the case of bone, as observed with other cells and tissue, the relationship of growth and differentiation must be maintained and stringently regulated, both during development and throughout the life of the organism, to support tissue remodeling. For many years, bone was defined anatomically and examined largely in a descriptive manner by ultrastructural analysis and by biochemical and histochemical methods. These studies provided the basis for our understanding of bone tissue organization and orchestration of the progressive recruitment, proliferation, and differentiation of the various cellular components of bone tissue. Now, complemented by an increased knowledge of molecular mechanisms that are associated with and regulate expression of genes encoding phenotypic compone...

Molecular mechanism mediating proliferation, defferentiation interralationships during progressie development of the osteoblast phenotype

Over the last decade the identification of mutations in the receptors for fibroblast growth factors (FGFs) has defined essential roles for FGF signaling in both endochondral and intramembranous bone development. FGF signaling pathways are essential for the earliest stages of limb development and throughout skeletal development. In this review, we examine the role of FGF signaling in bone development and in human genetic diseases that affect bone development. We also explore what is presently known about how FGF signaling pathways interact with other major signaling pathways that regulate chondrogenesis and osteogenesis. Overview of skeletal development Skeletal elements are formed through two distinct developmental processes. Endochondral ossification gives rise to long bones that comprise the appendicular skeleton, facial bones, vertebrae, and the lateral medial clavicles. Intramembranous ossification gives rise to the flat bones that comprise the cranium and medial clavicles. Both types of ossification involve an initial condensation of mesenchyme and the eventual formation of calcified bone. However, intramembranous bone formation accomplishes this directly, whereas endochondral ossification incorporates an intermediate step in which a cartilaginous template regulates the growth and patterning of the developing skeletal element. Development of endochondral bones initiates shortly after the formation of the limb bud with the condensation of loose mesenchyme, marked by expression of type

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FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease.

Skeletal homeostasis is determined by systemic hormones and local factors. Bone morphogenetic proteins (BMP) are unique because they induce the differentiation of mesenchymal cells toward cells of the osteoblastic lineage and also enhance the differentiated function of the osteoblast. However, the activity of BMPs needs to be tempered by intracellular and extracellular antagonists. BMPs bind to specific receptors and signal by phosphorylating the cytoplasmic proteins mothers against decapentaplegic (Smad) 1 and 5, which form heterodimers with Smad 4, and after nuclear translocation regulate transcription. BMP antagonists can be categorized as pseudoreceptors that compete with signaling receptors, inhibitory Smads that block signaling, intracellular binding proteins that bind Smad 1 and 5, and factors that induce ubiquitination and proteolysis of signaling Smads. In addition, a large number of extracellular proteins that bind BMPs and prevent their binding to signaling receptors have emerged. They are the ...

Bone morphogenetic proteins, their antagonists, and the skeleton.

The matrix metalloproteinases (MMPs) form an enzyme family of which gelatinase B (MMP-9) represents the largest and most complex member. We focus here on the biochemical properties, regulation, and functions of gelatinase B. The tight regulation of gelatinase B activity is highly complex and is established at five different levels. The transcription of the gelatinase B-gene depends on various cis-elements in its gene promotor and is induced or repressed by a large variety of soluble factors, including cytokines, growth factors, and hormones and by cellular contacts acting through specific signaling pathways. The specific regulation of its secretion occurs in cells storing gelatinase B in granules. After secretion, progelatinase B must be activated through an activation network. The enzyme activity is further regulated by inhibition and by other mechanisms, such as fine-tuning and stabilization by glycosylation. The ability of gelatinase B to degrade components of the extracellular matrix and to regulate the activity of a number of soluble proteins confers an important role in various physiological and pathological processes. These include reproduction, growth, development, inflammation, and vascular and proliferative diseases.

Biochemistry and Molecular Biology of Gelatinase B or Matrix Metalloproteinase-9 (MMP-9)

GH and IGF-I are important regulators of bone homeostasis and are central to the achievement of normal longitudinal bone growth and bone mass. Although GH may act directly on skeletal cells, most of its effects are mediated by IGF-I, which is present in the systemic circulation and is synthesized by peripheral tissues. The availability of IGF-I is regulated by IGF binding proteins. IGF-I enhances the differentiated function of the osteoblast and bone formation. Adult GH deficiency causes low bone turnover osteoporosis with high risk of vertebral and nonvertebral fractures, and the low bone mass can be partially reversed by GH replacement. Acromegaly is characterized by high bone turnover, which can lead to bone loss and vertebral fractures, particularly in patients with coexistent hypogonadism. GH and IGF-I secretion are decreased in aging individuals, and abnormalities in the GH/IGF-I axis play a role in the pathogenesis of the osteoporosis of anorexia nervosa and after glucocorticoid exposure.

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Growth Hormone, Insulin-Like Growth Factors, and the Skeleton

Insulin-like growth factor-I (IGF-I), a prevalent growth factor secreted by bone cells, has important effects on bone remodeling. Hormones are known to regulate the synthesis of skeletal IGF-I, but there is limited information about the actions of growth factors on IGF-I synthesis. We tested the effects of basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF beta 1), and platelet-derived growth factors (PDGF) AA and BB on IGF-I mRNA expression and polypeptide concentrations in cultures of osteoblast-enriched (Ob) cells from 22-day-old fetal rat calvariae. Steady state IGF-I mRNA levels were determined by Northern blot analysis, and IGF-I concentrations were determined in acidified and fractionated culture medium by a specific RIA. Treatment of Ob cells with bFGF at 0.06-6 nM, TGF beta 1 at 0.04-4 nM, and PDGF BB at 0.3-3.3 nM caused a dose-dependent decrease in steady state IGF-I mRNA. A smaller effect was observed with PDGF AA. The effect was initially observed after 6-8 h of treatment and was maximal after 16 h. Treatment with bFGF at 0.6-6 nM, TGF beta 1 at 0.4-4 nM, and PDGF BB at 0.3-3.3 nM for 24 h decreased IGF-I polypeptide concentrations by 40-80%. The effects of bFGF, TGF beta 1, and PDGF BB and AA on IGF-I mRNA were independent of protein synthesis and cell division, as they were observed in the presence and absence of cycloheximide at 3.6 microM or hydroxyurea at 1 mM. Similarly, their inhibitory actions on immunoreactive IGF-I were not prevented by hydroxyurea. In conclusion, bFGF, TGF beta 1, PDGF BB, and, to a lesser extent, PDGF AA decrease skeletal IGF-I synthesis by reducing IGF-I transcript levels, and this effect may contribute to their actions on selected aspects of Ob cell function.

Growth factors regulate the synthesis of insulin-like growth factor-I in bone cell cultures

Insulin-like growth factor-I (IGF-I) and IGF-II are among the most prevalent growth factors secreted by bone cells and are presumed to act as autocrine regulators of bone formation. We recently demonstrated that IGFs inhibit bone collagen degradation, and we postulated that they may either inhibit the expression of interstitial collagenase or stimulate the synthesis of tissue inhibitors of metalloproteinase-1 (TIMP-1), -2, or -3. We tested the effects of IGF-I and -II on collagenase and TIMP-1, -2, and -3 expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Steady state messenger RNA (mRNA) levels were determined by Northern blot analysis, and collagenase concentrations were determined in the culture medium by a specific immunoassay. After 2-6 h of treatment, IGF-I and -II decreased collagenase transcripts by up to 80%. IGF-I was a more potent inhibitor than IGF-II, because it was active at doses as low as 10 nM, whereas a dose of 100 nM was required to obser...

Insulin-like growth factors inhibit interstitial collagenase synthesis in bone cell cultures

Transforming growth factor (TGF) β1 is an autocrine regulator of bone cell function. We demonstrated that TGF β1 enhances bone collagen synthesis, but its effects on collagen degradation are not well characterized. We tested the effects of TGF β1 on rat collagenase 3 expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). Treatment with TGF β1 at 0.4 nM decreased steady state collagenase mRNA levels after 2 to 24 h. This dose-dependent effect was observed at TGF β1 concentrations of 4 pM to 1.2 nM, and was accompanied by decreased levels of immunoreactive procollagenase. The protein synthesis inhibitor cycloheximide increased collagenase transcripts, but did not prevent the effect of TGF β1 on collagenase mRNA levels. TGF β1 accelerated the decay of collagenase mRNA in transcriptionally arrested Ob cells. In addition, TGF β1 decreased the levels of collagenase heterogeneous nuclear RNA and the rate of collagenase gene transcription in Ob cells. TGF β1 enhanced the expression of tissue inhibitors of metalloproteinases (TIMP) 1 and 3 and caused a modest decrease of TIMP 2 mRNA levels. In conclusion, TGF β1 decreases interstitial collagenase transcripts and protease levels in Ob cells by transcriptional and post-transcriptional mechanisms, and this effect may contribute to its actions on bone matrix. J. Cell. Physiol. 170:145–152, 1997. © 1997 Wiley-Liss, Inc.

Transforming growth factor β1 inhibits collagenase 3 expression by transcriptional and post‐transcriptional mechanisms in osteoblast cultures

Basic fibroblast growth factor (bFGF) is a bone cell mitogen that affects osteoblastic function by suppressing type I collagen synthesis. The investigators in this study examined whether bFGF also regulates interstitial collagenase and tissue inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells isolated from 22-day fetal rat calvariae. After exposure to 600 pM bFGF, interstitial collagenase messenger RNA (mRNA) levels, as determined by Northern hybridization analysis, increased after 2 h and were maximally stimulated to approximately 13-fold at 6 h. Exposure of osteoblast-enriched cells to 0.06-6 nM bFGF increased collagenase mRNA in a dose-dependent manner, and bFGF also increased immunoreactive collagenase measured in the culture medium by Western blot analysis. The protein synthesis inhibitor cycloheximide, as well as two inhibitors of protein kinase C, staurosporine and sangivamycin, prevented the bFGF induction of collagenase transcripts, whereas indomethacin, an inhibitor of prostag...

Basic fibroblast growth factor stimulates expression of interstitial collagenase and inhibitors of metalloproteinases in rat bone cells.

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor superfamily of peptides, induces ectopic bone formation in vivo. The actions of BMP-2 on osteoblastic cells include stimulation of collagen synthesis, but the role of BMP-2 on collagen degradation is not known. We examined whether BMP-2 affects the expression of collagenase-3, an enzyme that degrades type I collagen at neutral pH, and that of tissue inhibitors of matrix metalloproteinases (TIMPs) in primary osteoblast-enriched cells from 22-day-old fetal rat calvariae. BMP-2 suppressed collagenase messenger RNA (mRNA) and immunoreactive protein levels. BMP-2 did not affect collagenase mRNA stability, but it reduced collagenase heterogeneous nuclear RNA levels and decreased the rate of transcription of the collagenase gene. BMP-2 also stimulated TIMP 1 and TIMP 3 mRNA levels, but failed to alter TIMP 2 expression. In conclusion, our studies indicate that BMP-2 suppresses collagenase-3 gene transcription and stimulates TIMP 1 a...

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Samuel Varghese

Papers

Growth factors regulate the synthesis of insulin-like growth factor-I in bone cell cultures

Insulin-like growth factors inhibit interstitial collagenase synthesis in bone cell cultures

Transforming growth factor β1 inhibits collagenase 3 expression by transcriptional and post‐transcriptional mechanisms in osteoblast cultures

Basic fibroblast growth factor stimulates expression of interstitial collagenase and inhibitors of metalloproteinases in rat bone cells.

Regulation of Collagenase-3 by Bone Morphogenetic Protein-2 in Bone Cell Cultures