Jan O. Gordeladze

Leptin stimulates human osteoblastic cell proliferation, de novo collagen synthesis, and mineralization: Impact on differentiation markers, apoptosis, and osteoclastic signaling

Anabolic hormones, mechanical loading, and the obese protein leptin play separate roles in maintaining bone mass. We have previously shown that leptin, as well as its receptor, are expressed by normal human osteoblasts. Consequently, we have investigated how leptin affects proliferation, differentiation, and apoptosis of human osteoblasts. Iliac crest osteoblasts, incubated with either leptin (100 ng/ml), calcitriol (1,25(OH)2D3; 10−9 M) or 1–84 human parathyroid hormone (PTH; 10−8 M), were cultured for 35 consecutive days and assayed for expression of various differentiation‐related marker genes (as estimated by RT‐PCR), de novo collagen synthesis, proliferation, in vitro mineralization, and osteoclast signaling. The effects of leptin on protection against retinoic acid (RA; 10−7 M) induced apoptosis, as well as transition into preosteocytes, were also tested. Leptin exposure enhanced cell proliferation and collagen synthesis over both control condition and PTH exposure. Leptin inhibited in vitro calcified nodule production after 1–2 weeks in culture, however, subsequent to 4–5 weeks, leptin significantly stimulated mineralization. The mineralization profile throughout the entire incubation period was almost undistinguishable from the one induced by PTH. In comparison, 1,25(OH)2D3 generally reduced proliferation and collagen production rates, whereas mineralization was markedly enhanced. Leptin exposure (at 2 and 5 weeks) significantly enhanced the expression of TGFβ, IGF‐I, collagen‐Iα, ALP, and osteocalcin mRNA. Leptin also protected against RA‐induced apoptosis, as estimated by soluble DNA fractions and DNA laddering patterns subsequent to 10 days of culture. The expression profiles of Bax‐α and Bcl‐2 mRNAs indicated that leptin per se significantly protected against apoptosis throughout the entire incubation period. Furthermore, the osteoblast marker OSF‐2 was diminished, whereas the CD44 osteocyte marker gene expression was stimulated, indicating a transition into preosteocytes. In terms of osteoclastic signaling, leptin significantly augmented the mRNA levels of both interleukin‐6 (IL‐6) and osteoprotegerin (OPG). In summary, continuous leptin exposure of iliac crest osteoblasts, promotes collagen synthesis, cell differentiation and in vitro mineralization, as well as cell survival and transition into preosteocytes. Leptin may also facilitate osteoblastic signaling to the osteoclast.

Leptin Is Expressed in and Secreted from Primary Cultures of Human Osteoblasts and Promotes Bone Mineralization

The adipose hormone leptin and its receptor are important for regulation of food intake and energy metabolism. Leptin also is involved in the growth of different tissues. In this study, we show the expression of leptin in primary cultures of normal human osteoblasts (hOBs) as evidenced by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunocytochemistry. Release of leptin into the medium also was found. Leptin was not detected in commercially available hOBs (NHOst) or in three different human monoclonal osteosarcoma cell lines. Leptin expression was observed in OBs in the mineralization and/or the osteocyte transition period but not during the matrix maturation period. Furthermore, hOBs and osteosarcoma cell lines expressed the long signal‐transducing form of the leptin receptor (OB‐Rb) as shown by RT‐PCR. We observed no significant changes in leptin or OB‐Rb genes in hOBs after incubation with recombinant leptin, indicating no autoregulation of the leptin expression. Incubation of both hOBs entering the mineralization phase and osteosarcoma cell lines with recombinant leptin markedly increased the number of mineralized nodules as shown by alizarin S staining. These findings indicate that leptin may be of importance for osteoblastic cell growth and bone mineralization.

Cartilage Tissue Engineering: Towards a Biomaterial-Assisted Mesenchymal Stem Cell Therapy

Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, the lack of efficient modalities of treatment for large chondral defects has prompted research on tissue engineering combining chondrogenic cells, scaffold materials and environmental factors. The aim of this review is to focus on the recent advances made in exploiting the potentials of cell therapy for cartilage engineering. These include: 1) defining the best cell candidates between chondrocytes or multipotent progenitor cells, such as multipotent mesenchymal stromal cells (MSC), in terms of readily available sources for isolation, expansion and repair potential; 2) engineering biocompatible and biodegradable natural or artificial matrix scaffolds as cell carriers, chondrogenic factors releasing factories and supports for defect filling, 3) identifying more specific growth factors and the appropriate scheme of application that will promote both chondrogenic differentiation and then maintain the differentiated phenotype overtime and 4) evaluating the optimal combinations that will answer to the functional demand placed upon cartilage tissue replacement in animal models and in clinics. Finally, some of the major obstacles generally encountered in cartilage engineering are discussed as well as future trends to overcome these limiting issues for clinical applications.

A unified model for the action of leptin on bone turnover

Leptin has been advocated as a centrally acting factor responsible for inhibiting accumulation of bone mass. However, recent investigations unequivocally establish leptin as a local (autocrine) factor expressed by osteoblasts. Exogenously added leptin causes osteoblastic cell proliferation and differentiation, while also rendering osteoblasts more efficacious in terms of mineralization. Leptin acts as an anti‐apoptotic agent, and augments messages responsible for the remodelling of bone tissue, i.e., mRNAs for osteoprotegerin (OPG) and the interleukin IL‐6. Furthermore, leptin message is readily expressed in osteoblasts subjected to mechanical strain. In this respect, osteoblasts, which are unilaterally stretched proliferate and differentiate, a phenomenon being potentiated by exposure of the cells to differentiating humoral factors. This article discusses a unified model of dually acting leptin through the central nervous system and the mechanostat principle applied to osteoblasts. The proposed model may account for the finely tuned bone homeostasis maintained within rather narrow limits, depending on exposure to humoral factors and the prevailing mechanostat usage mode.

Vasoactive intestinal peptide and peptide with N-terminal histidine and C-terminal isoleucine increase prolactin secretion in cultured rat pituitary cells (GE4C1) via a cAMP-dependent mechanism which involves transient elevation of intracellular Ca2+

Vasoactive intestinal peptide (VIP) and peptide (P) with N-terminal histidine and C-terminal isoleucine (PHI) stimulated prolactin (PRL) secretion from GH4C1 cells equipotent with ED50 values of 30-50 nM. In a parafusion system optimized to give high time resolution both VIP and PHI increased PRL secretion with a delay of about 60 s and subsequent to the activation of the adenylate cyclase. Thyroliberin (TRH) increased PRL secretion within 4 s. The dose-response curves for VIP- and PHI-stimulated cAMP accumulation were superimposable on those for PRL secretion. At submaximal concentrations the effects of VIP and PHI on both cAMP accumulation and PRL secretion were additive, whereas the effects were not additive at concentrations giving maximal effects. VIP and PHI increased [Ca2+]i measured by quin-2 in a different way than TRH, without inducing changes in the electrophysiological membrane properties of the GH4C1 cells. We conclude that both VIP and PHI stimulate PRL secretion via a cAMP-dependent process involving an increase in [Ca2+]i.

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.

Role of leptin in bone growth: central player or peripheral supporter?

Maintenance or gain of bone mass is associated with hormone and/or heavy mechanical load, either due to obesity or physical activity [1]. Amongst the many hormones that might account for the link between energy and bone metabolism is leptin. In this respect, much attention has focused on the e¡ects of leptin as a central satiety agent, although in vitro studies provide evidence for direct e¡ects on speci¢c tissues and metabolic pathways. The debate related to central and/or peripheral regulation of bone metabolism by leptin has been ongoing during the last 2 years. The article by Lee and colleagues [2] in this issue further addresses this topic, and provides evidence that functional leptin receptors are expressed in rat osteoblasts. These results point towards a more direct role of leptin in bone growth. Since its discovery in 1994 [3], leptin has been acknowledged as an adipocyte-derived signaling molecule, which may limit food intake and increase energy expenditure via speci¢c receptors located in the central nervous system. However, the picture is becoming more complicated, and leptin is now looked upon as a multi-potent cytokine with both indirect central and direct peripheral e¡ects in di¡erent organs, tissues and cells. Circulating leptin in adult individuals is mainly secreted from adipose tissue [3,4], although leptin polypeptide and mRNA are detected in other tissues like muscle [5], gastric epithelium [6], bone [7], and in breast epithelial cells [8]. Leptin expression has also been found in arterial wall cells [9] and in normal pituitary and di¡erent types of pituitary adenomas [10]. In pregnant women, leptin is synthesized and secreted from placental trophoblasts into the maternal and fetal circulation [11]. Leptin binds to receptors in the hypothalamus [4] which leads to reduced appetite and increased energy expenditure. The leptin receptor (OB-R) exhibits considerable homology with the interleukin-6 (IL-6) receptor and belongs to the cytokine class I receptor family [12]. At least six forms of the leptin receptor have been found, of which the long signaltransducing form, OB-RL, presumably mediates most of leptin’s signaling events. It is also the isoform most abundantly expressed in the hypothalamus [12]. However, the leptin receptor is expressed in various areas of the central nervous system, and in many other organs and cell types [13]. The cytokine receptor superfamily is a rapidly growing family of receptors. An early and most likely pivotal event for all subspecies is the activation of one or more members of the Janus (or JAK) family of tyrosine kinases. The activated JAK kinases, which form a complex with the cytokine receptor subunits, induce autophosphorylation as well as phosphorylation of the receptor. These phosphorylated tyrosines form binding sites for various signaling molecules that are themselves thought to be phosphorylated by JAK kinases, including signal transducers and activators of transcription (STATs) [14], which regulate transcription by adapter proteins like SH2-containing protein tyrosine phosphatase [15] that recruit Grb2 complexes thereby initiating the mitogen-activated protein kinase (MAPK) pathway, and insulin receptor substrate (IRS) proteins that, through phosphate inositol pathway, are thought to regulate metabolic events in the cell [16].

Concerted stimuli regulating osteo-chondral differentiation from stem cells: phenotype acquisition regulated by microRNAs.

AbstractBone and cartilage are being generated de novo through concerted actions of a plethora of signals. These act on stem cells (SCs) recruited for lineage-specific differentiation, with cellular phenotypes representing various functions throughout their life span. The signals are rendered by hormones and growth factors (GFs) and mechanical forces ensuring proper modelling and remodelling of bone and cartilage, due to indigenous and programmed metabolism in SCs, osteoblasts, chondrocytes, as well as osteoclasts and other cell types (eg T helper cells).This review focuses on the concerted action of such signals, as well as the regulatory and/or stabilizing control circuits rendered by a class of small RNAs, designated microRNAs. The impact on cell functions evoked by transcription factors (TFs) via various signalling molecules, also encompassing mechanical stimulation, will be discussed featuring microRNAs as important members of an integrative system. The present approach to cell differentiation in vitro may vastly influence cell engineering for in vivo tissue repair.

Cell specific distribution of guanine nucleotide-binding regulatory proteins in rat pituitary tumour cell lines.

To investigate the effects of guanine nucleotide-binding regulatory proteins (G proteins) on hormonal regulation of prolactin (PRL) synthesis and secretion, the qualitative distribution of G protein alpha-subunits and their mRNAs was studied in three functionally different pituitary tumour cell lines (GH cells) and normal rat pituitary tissue. Levels of basal and modulated adenylyl cyclase (AC) and phospholipase C (PLC) activities are also included. GH cells and pituitary tissue contained various amounts of mRNAs and protein for Gs alpha, Gi-2 alpha, Gi-3 alpha and Go alpha, while mRNA for Gi-1 alpha was only detected in normal pituitary tissue. Gz alpha/Gx alpha mRNA was expressed in all pituitary cell lines as well as in pituitary tissue. Go alpha mRNA and Gz alpha/G x alpha mRNA displayed size heterogeneity. These findings may have importance in the understanding of hormone regulation of second messenger systems.

Stage Dependent Variation in Mn2+−-Sensitive Adenylyl Cyclase (AC) Activity in Spermatids and FSH-Sensitive AC in Sertoli Cells

The variation of the specific Mn2+-dependent adenylyl cyclase (AC activity in spermatids and follicle stimulating hormone (FSH)-responsive AC activities in Sertoli cells in different stages (I-XIV) of the seminiferous epithelial cycle has been investigated. Maximal Mn2+-dependent AC activity was observed in stages II-III while minimal activity was encountered in stages VII-VIII (spermiation). FSH-responsive AC activity exhibited a pattern that coincided with that of the Mn2+-dependent AC. The stage-dependent variation in spermatid AC activity cannot be explained by altered numbers of haploid cells. This raises the question whether the Sertoli cells may regulate the spermatid AC activity. Sertoli cells in various stages are all exposed to the same concentration of circulatory hormones. Hence the stage-dependent difference in FSH-responsiveness indicates that local influences (from germ cells?) may regulate the response of the AC in Sertoli cells to FSH.