BMPR1B

LSJL upregulates BMPR1B.


Impairment of Sox9 expression in limb buds of rats homozygous for hypodactyly mutation.

"Rat hypodactyly (hd) is an autosomal recessive mutation manifesting in homozygotes as reduction or loss of digits II and III. We mapped the hd allele to a short segment of chromosome 10, containing 16 genes. None of these genes has been shown to influence limb development yet. In situ hybridization showed no changes in several important patterning genes (Shh, Fgf8, Bmp2, 4, 7). However, we found that expression of cartilage condensation marker Sox9, and Bmp receptor Bmpr1b (acting as an upstream activator of Sox9 expression) is absent from the subepithelial mesenchyme of the digit condensations II and III. The failure of the chondrogenic condensations to extend towards the subepithelial mesenchyme may reduce the size of digit primordia and underlie the subsequent loss of phalanges and reduction of metacarpals/metatarsals in hd rats."

"Bmpr1a conditional knockout mice exhibit generalized chondrodysplasia with long bones shortened and ossification delayed. In double (Bmpr1a-/- and Bmpr1b-/-) mutants, all bones that form through endochondral ossification are either absent or malformed, and differentiation of prechondrocytes into chondrocytes is severely affected. Importantly, double mutants do not express Sox9, indicating that BMP signalling acts as an upstream activator of Sox9 expression."

Pax1

Pax1 and Pax9 activate Bapx1 to induce chondrogenic differentiation in the sclerotome

"We have previously shown that the paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. We show, by analyzing Pax1;Pax9 double mutant mice, that expression of Bapx1[Also known as Nkx3.2] in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, we show that Pax1 can substitute for Shh in inducing Bapx1 expression and in initiating chondrogenic differentiation. Furthermore, we demonstrate that Pax1 and Pax9 can transactivate regulatory sequences in the Bapx1 promoter and that they physically interact with the Bapx1 promoter region. These results strongly suggest that Bapx1 is a direct target of Pax1 and Pax9. Together, we conclude that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells."

Tall with Vimentin?

The intermediate filament vimentin regulates chondrogenesis of adult human bone marrow-derived multipotent progenitor cells.

"Actin and tubulin [regulate] developmental chondrogenesis. Vimentin [regulates[ the chondrogenic differentiation of adult multipotent progenitor cells (MPCs). As our model of adult progenitor cell chondrogenesis, we employed high-density pellet cultures of human bone marrow-derived MPCs. siRNA-mediated knockdown of vimentin mRNA and protein triggered a reduction in the extent of MPC cartilage formation, as evidenced by depressed accumulation of mRNAs for the cartilage-specific marker genes aggrecan and collagen type II, as well as reduced levels of Alcian blue-stainable proteoglycan and collagen II protein in the extracellular matrix. Moreover, mRNA and protein levels for the chondro-regulatory transcription factors SOX5, SOX6, and SOX9 were diminished by vimentin knockdown. Depleted cellular vimentin also induced a drastic reduction in PKA phosphorylation levels but did not affect the phosphorylation of multiple other chondro-regulatory kinases and transcription factors, including ERK1/2, p38, Smad2, and Smad1/5/8. Importantly, siRNA-mediated knockdown of PKA C-alpha mRNA and protein mimicked the reduction in chondrogenesis caused by diminished cellular vimentin. Finally, overexpression of vimentin in MPCs significantly enhanced the activity of a transfected collagen II promoter-luciferase reporter gene."

"actin transduces the chondro-inhibitory effects of integrin adhesion in bovine bone marrow-derived mesenchymal stem cells (MSCs) cultured in an agarose hydrogel three-dimensional environment"

"dedifferentiated chondrocytes can be stimulated to re-express genes encoding cartilage-specific ECM molecules via cytochalasin-induced actin disruption"

"depleted cellular vimentin is associated with significantly depressed PKA phosphorylation levels and correspondingly that siRNA-mediated knockdown of PKA C-α mRNA and protein mimics the chondro-inhibitory effects of vimentin depletion. Finally, we found that vimentin overexpression markedly enhances adult human MPC pellet culture chondrogenesis."

"PKA C-α knockdown decreased AGC, COL2A1, SOX9, L-SOX5, and SOX6 mRNA transcript levels relative to negative controls"

"A plethora of intracellular signaling pathways have been implicated in the regulation of cartilage formation in embryonic chondro-progenitor cells, chondrogenic cell lines, and adult MSCs, including the Smad1/5/8 and 2/3 pathways, as well as the PKA, ERK1/2, p38, and JNK cascades"<-LSJL involves Smad1/5/8, ERK1/2, and P38.

ADAMTS

LSJL upregulates ADAMTS 1,4, and downregulates 7.

Regulation of aggrecanases from the ADAMTS family and aggrecan neoepitope formation during in vitro chondrogenesis of human mesenchymal stem cells.

"Aggrecanases from the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family are important therapeutic targets due to their essential role in aggrecan depletion in arthritic diseases. Whether their function is also important for matrix rearrangements during chondrogenesis and thus, cartilage regeneration, is however so far unknown. The aim of this study was to analyse the expression and function of ADAMTS with aggrecanase activity during chondrogenic differentiation of human mesenchymal stem cells (MSCs). Chondrogenic differentiation was induced in bone marrow-derived MSC pellets and expression of COL2A1, aggrecan, ADAMTS1, 4, 5, 9, 16 and furin was followed by quantitative RT-PCR. Formation of the NITEGE (ADAMTS-cleaved) and DIPEN (MMP-cleaved) aggrecan neoepitopes was detected by immunohistochemistry. While the expression of ADAMTS4, 9, 16 and furin was up-regulated during chondrogenesis, ADAMTS1 and 5 were down-regulated. Despite this regulation of ADAMTS, no formation of NITEGE neoepitopes occurred in MSC pellets, indicating no ADAMTS-induced cleavage of aggrecan. In contrast, MMP-induced cleavage of aggrecan appeared at 14 d after induction of chondrogenesis. Submission of differentiated MSC pellets to IL1β treatment for 3 d resulted in strong upregulation of ADAMTS1, 4 and 5, rapid proteoglycan depletion, and stimulation of ADAMTS-induced but not MMP-induced cleavage of aggrecan. Thus, there is no evidence for ADAMTS-induced aggrecan cleavage during chondrogenesis, but proteoglycan turnover is rapidly inducible under inflammatory signals. "

"ADAMTS induced cleavage of ACAN may not be implicated in chondrogenic differentiation and that therefore the chondroprotective effect of aggrecanase inhibitors may not be associated with negative effects on chondrogenic and regenerative processes."

Activin RII and LSJL

LSJL upregulates Activin RII.


Immunohistochemical detection of activin A, follistatin, and activin receptors during fracture healing in the rat.

"Activins exert their cellular effects by way of activin type-I and type-II serine/threonine kinase receptors. Follistatin is an activin-binding protein that can suppress the biological effects of activins. In this study, the immunohistochemical expression of activin A, follistatin, and activin receptors was studied during fracture healing in the rat. Activin A was weakly detected in the periosteum near the fracture ends at an early stage but was absent in the chondrocytes around the fracture gap, where endochondral ossification took place. An antibody to follistatin stained osteogenic cells in the periosteum near the fracture ends; moderate and strong staining were observed in proliferating, mature, and hypertrophied chondrocytes at the sites of endochondral ossification. Levels of activin A and follistatin were high near the osteoblasts on the surface of the newly formed trabecular bone[The increase in FST in LSJL could be a result of new trabecular bone formation]. In addition, an intense localization of activin A was noted where multinucleated osteoclast-like cells were present. This study suggests that the activin-follistatin system may contribute to cellular events related to the formation and remodeling of bone during fracture healing. Activin type-I and type-II receptors were co-expressed in intramembranous and endochondral ossification sites[or it could be this]. The expression of activin type-I, type-II, and type-IIB receptors in the absence of activin A in the endochondral ossification suggests that other isoforms of activins may signal by way of these receptors."

"certain BMPs were shown to bind to activin receptors as well as to their own BMP receptors"

"In normal intact femurs of the rat, activin type-IB and type-II-but not type-I-receptors were weakly expressed in osteogenic cells at the periosteum; however, activin type-I and type-I1 receptors were not observed in osteocytes at the cortical bone."

BMP-3 signals through Activin RII.

Myostatin inhibition induces muscle fibre hypertrophy prior to satellite cell activation., states that Activin RII can inhibit Myostatin.

bisphosphonate

COMPARISON OF BISPHOSPHONATE TREATMENT REGIMENS ON SKELETAL GROWTH & BIOMECHANICS

"Bisphosphonates have been shown to be safe and effective for the treatment of disorders associated with increased bone resorption in humans. However, these studies have focused on adults in whom the growth plate is fused, thus there is no longitudinal bone growth and little/no appositional bone growth. Although bisphosphonate administration in the pediatric population was initially pioneered for compassionate use in children with severe Osteogenesis Imperfecta, these medications are being increasingly used for other disorders, ranging in severity from the prevention of steroid-induced osteoporosis in ambulatory children affected with connective tissue, gastrointestinal and pulmonary diseases, to prevention of bone loss in children with hypercalciuria. Hypophosphatemia impairs apoptosis of hypertrophic chondrocytes, both in vivo and in vitro, leading to the development of rickets in growing animals. The nitrogen-containing bisphosphonate, alendronate, prevents phosphate-mediated apoptosis of hypertrophic chondrocytes in vitro. In vivo studies in bisphosphonate-treated mice and rabbits demonstrate decreased long bone growth, accompanied by expansion of the hypertrophic chondrocyte layer and retention of cartilage in cortical bone. The studies in this proposal will examine the effects of bisphosphonates on growing bone. They will address the hypothesis that select bisphosphonates impair hypertrophic chondrocyte apoptosis in vitro and in vivo and attenuate vascular invasion of the maturing chondro-osseous junction. They will also address the hypothesis that for some agents, the doses required for prevention of bone loss will not parallel that which adversely effects the growth plate. The structural characteristics, tissue properties and biomechanical integrity of the skeleton of bisphosphonate treated mice will be examined to address whether the accrual of metaphyseal bone after treatment discontinuation, which has lower density than bone accrued during treatment, and/or the presence of cartilage in cortical bone, result in zones of localized bone fragility leading to impaired biomechanical integrity. Investigations will be performed with nitrogen and non-nitrogen containing bisphosphonates and will examine the consequences of acute and chronic bisphosphonate administration using different administration schedules (daily, bi-weekly or every other month). While these investigations are not expected to change the use of bisphosphonates as the first line of therapy for patients with severe Osteogenesis Imperfecta, they are expected to impact on the pharmacological agent and delivery method selected to preserve bone mass, biomechanical integrity of bone and longitudinal growth in children with disorders that require bisphosphonate treatment to minimize skeletal morbidity."