Members of the transforming growth factor beta (TGFβ) superfamily of secreted factors play essential roles in nearly every aspect of cartilage formation and maintenance. However, the mechanisms by which TGFβs transduce their effects in cartilage in vivo remain poorly understood. Mutations in several TGFβ family members, their receptors, extracellular modulators, and intracellular transducers have been described, and these usually impact the development of the cartilaginous skeleton. Furthermore, genome-wide association studies have linked components of the (TGFβ) superfamily to susceptibility to osteoarthritis. This review focuses on recent discoveries from genetic studies in the mouse regarding the regulation of TGFβ signaling in developing growth plate and articular cartilage, as well as the different modes of crosstalk between canonical and noncanonical TGFβ signaling. These new insights into TGFβ signaling in cartilage may open new prospects for therapies that maintain healthy articular cartilage.

The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major method for detecting changes in skeletal patterning. Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively.

Bone morphogenetic proteins (BMPs) are crucial regulators of chondrogenesis. BMPs transduce their signals through three type I receptors: BMPR1A, BMPR1B, and ACVR1/ALK2. Fibrodysplasia ossificans progressiva (FOP), a rare disorder characterized by progressive ossification of connective tissue, is caused by an activating mutation in Acvr1 (the gene that encodes ACVR1/ALK2). However, there are few developmental defects associated with FOP. Thus, the role of ACVR1 in chondrogenesis during development is unknown. Here we report the phenotype of mice lacking ACVR1 in cartilage. Acvr1CKO mice are viable but exhibit defects in the development of cranial and axial structures. Mutants exhibit a shortened cranial base, and cervical vertebrae are hypoplastic. Acvr1CKO adult mice develop progressive kyphosis. These morphological defects were associated with decreased levels of Smad1/5 and p38 activation, and with reduced rates of chondrocyte proliferation in vertebral cartilage. We also tested whether ACVR1 exerts coordinated functions with BMPR1A and BMPR1B through analysis of double mutants. Acvr1/Bmpr1a and Acvr1/Bmpr1b mutant mice exhibited generalized perinatal lethal chondrodysplasia that was much more severe than in any of the corresponding mutant strains. These findings demonstrate that ACVR1 is required for chondrocyte proliferation and differentiation, particularly in craniofacial and axial elements, but exerts coordinated functions with both BMPR1A and BMPR1B throughout the developing endochondral skeleton.

Spondylocarpotarsal synostosis (SCT) is an autosomal recessive disorder characterized by progressive vertebral fusions and caused by loss of function mutations in Filamin B (FLNB). FLNB acts as a signaling scaffold by linking the actin cytoskleteon to signal transduction systems, yet the disease mechanisms for SCT remain unclear. Employing a Flnb knockout mouse, we found morphologic and molecular evidence that the intervertebral discs (IVDs) of Flnb–/–mice undergo rapid and progressive degeneration during postnatal development as a result of abnormal cell fate changes in the IVD, particularly the annulus fibrosus (AF). In Flnb–/–mice, the AF cells lose their typical fibroblast-like characteristics and acquire the molecular and phenotypic signature of hypertrophic chondrocytes. This change is characterized by hallmarks of endochondral-like ossification including alterations in collagen matrix, expression of Collagen X, increased apoptosis, and inappropriate ossification of the disc tissue. We show that conversion of the AF cells into chondrocytes is coincident with upregulated TGFβ signaling via Smad2/3 and BMP induced p38 signaling as well as sustained activation of canonical and noncanonical target genes p21 and Ctgf. These findings indicate that FLNB is involved in attenuation of TGFβ/BMP signaling and influences AF cell fate. Furthermore, we demonstrate that the IVD disruptions in Flnb–/–mice resemble aging degenerative discs and reveal new insights into the molecular causes of vertebral fusions and disc degeneration.

Osteoporosis is a disease characterized by low bone mass, leading to an increased risk of fragility fractures. GATA4 is a zinc-finger transcription factor that is important in several tissues, such as the heart and intestines, and has recently been shown to be a pioneer factor for estrogen receptor alpha (ERα) in osteoblast-like cells. Herein, we demonstrate that GATA4 is necessary for estrogen-mediated transcription and estrogen-independent mineralization in vitro. In vivo deletion of GATA4, driven by Cre-recombinase in osteoblasts, results in perinatal lethality, decreased trabecular bone properties, and abnormal bone development. Microarray analysis revealed GATA4 suppression of TGFβ signaling, necessary for osteoblast progenitor maintenance, and concomitant activation of BMP signaling, necessary for mineralization. Indeed, pSMAD1/5/8 signaling, downstream of BMP signaling, is decreased in the trabecular region of conditional knockout femurs, and pSMAD2/3, downstream of TGFβ signaling, is increased in the same region. Together, these experiments demonstrate the necessity of GATA4 in osteoblasts. Understanding the role of GATA4 to regulate the tissue specificity of estrogen-mediated osteoblast gene regulation and estrogen-independent bone differentiation may help to develop therapies for postmenopausal osteoporosis.