Arunik Sanyal

Role of RANK ligand in mediating increased bone resorption in early postmenopausal women

Studies in rodents have implicated various cytokines as paracrine mediators of increased osteoclastogenesis during estrogen deficiency, but increases in RANKL, the final effector of osteoclastogenesis, have not been demonstrated. Thus, we isolated bone marrow mononuclear cells expressing RANKL on their surfaces by two-color flow cytometry using FITC-conjugated osteoprotegerin-Fc (OPG-Fc-FITC) as a probe. The cells were characterized as preosteoblastic marrow stromal cells (MSCs), T lymphocytes, or B lymphocytes by using Abs against bone alkaline phosphatase (BAP), CD3, and CD20, respectively, in 12 premenopausal women (Group A), 12 early postmenopausal women (Group B), and 12 age-matched, estrogen-treated postmenopausal women (Group C). Fluorescence intensity of OPG-Fc-FITC, an index of the surface concentration of RANKL per cell, was increased in Group B over Groups A and C by two- to threefold for MSCs, T cells, B cells, and total RANKL-expressing cells. Moreover, in the merged groups, RANKL expression per cell correlated directly with the bone resorption markers, serum C-terminal telopeptide of type I collagen and urine N-telopeptide of type I collagen, in all three cell types and inversely with serum 17beta-estradiol for total RANKL-expressing cells. The data suggest that upregulation of RANKL on bone marrow cells is an important determinant of increased bone resorption induced by estrogen deficiency.

An effective method of completely removing contaminating genomic DNA from an RNA sample to be used for PCR

A simple method for removing contaminating genomic DNA from an RNA preparation is presented. The method involves digestion of the RNA with RNase-free DNase I at room temperature followed by inactivation of the enzyme at 65°C in presence of EDTA. This method produces an RNA sample that is negative for genomic DNA by PCR.

The spatiotemporal expression of TGF-β1 and its receptors during periosteal chondrogenesis in vitro

Transforming growth factor‐β1 (TGF‐β1) has been shown to stimulate chondrogenesis in periosteal explants cultured in agarose suspension. TGF‐βs exert their cellular effects through a heteromeric cell membrane receptor complex consisting of TGF‐β type I and type II receptors. In this study, the spatial and temporal expressions of the type I receptor (TβR‐I), type II receptor (TβR‐II) and endogenous TGF‐β1 in periosteal explants cultured in vitro were examined using reverse transcription polymerase chain reaction (RT‐TCR) and immunohistochemistry. The temporal changes in the expression of the TβR‐I and TβR‐II mRNAs correlated with that of TGF‐β1. Exogenous administration of TGF‐β1 upregulated the expression of both receptors and of the TGF‐β1 ligand in a biphasic pattern. The earlier peak of upregulation was observed at 7 days in culture. A later peak of upregulation was seen at 42 days, at which time cartilage formation reached a maximum. Immunohistochemical studies demonstrated co‐localization and TβR‐II simultaneously among the same cells expressing TGF‐β1. TGF‐β1 treatment increased the expression of TGF‐β1, I and TβR‐II in mesenchymal cells in the cambium layer at 7 days in culture. Small round chondrocytes showed widely distributed immunoreactivity of TGF‐β1, TβR‐I and TβR‐II in the 42‐day explants treated with TGF‐β1. These observations support the hypothesis that TGF‐β1 regulates the initiation and formation of cartilage during periosteal chondrogenesis.

Histomorphological and proliferative characterization of developing periosteal neochondrocytes in vitro.

Periosteal chondrogenesis is relevant to cartilage repair and fracture healing. Cell proliferation is a limiting factor of cartilage production. We used an in vitro organ culture model to test the hypothesis that proliferative activity correlates with cell morphology. One hundred and four periosteal explants from 26 two‐month old New Zealand rabbits were cultured for up to 42 days. They were analyzed histomorphologically, and immunohistochemically with proliferative cell nuclear antigen (PCNA). The periosteal neocartilage displayed a consistent zonal pattern of chondrocyte cell shapes. The flat cell zone from day 7 to 21, consisted of uniform‐sized small spindle‐shaped cells. The round cell zone, which appeared on day 14, consisted of variable‐sized round cells averaging 510 ± 250 μm2 in area. They were subdivided into small round (<510 μm2) and large round cells (>510 μm2). The proliferative index was highest in the small round cell group (32 ± 6%), intermediate in the flat cell group (27 ± 6%), and lowest in the large round cell group (20 ± 7%) (P < 0.001). Furthermore, the proliferative indices in the round cell group were inversely proportional to cell size. Therefore, (1) there is a sequential progression of cell morphology during periosteal chondrogenesis, (2) cell differentiation is arrested prior to terminal differentiation for some cells and not for others, and (3) proliferative activity is strongly related to cell morphology. This organ culture model provides us with opportunities to study the regulation of terminal chondrocyte differentiation and the control of cell proliferation. This will contribute to our understanding of cartilage repair, fracture healing and growth plate physiology.

Regulation of Bone Turnover by Sex Steroids in Men

Introduction: The mechanism(s) by which sex steroids regulate bone turnover in humans are unclear, and recent studies have suggested that follicle‐stimulating hormone (FSH) may play an important role in regulating bone resorption.

A fragment of the hypophosphatemic factor, MEPE, requires inducible cyclooxygenase‐2 to exert potent anabolic effects on normal human marrow osteoblast precursors

MEPE, 56.6 kDa protein isolated from tumors associated with hypophosphatemic osteomalacia, increases renal phosphate excretion and is expressed in normal human bone cells. AC‐100, a central 23‐amino acid fragment of MEPE, contains motifs that are important in regulating cellular activities in the bone microenvironment. Thus, we assessed in vitro effects of AC‐100 on multipotential normal human marrow stromal (hMS) cells that have the capacity to differentiate into mature osteoblasts. Proliferation was quantified by [H3]thymidine uptake and cell counting and differentiation by the levels of mRNA for the α2‐chain of type I procollagen (COL1A2), alkaline phosphatase (AP), and osteocalcin (OC) measured using real time reverse transcriptase PCR (RT‐PCR) and by the formation of mineralized nodules. AC‐100 increased proliferation by 257 ± 89% (P < 0.005), increased gene expression of COL1A2 by 339 ± 85% (P < 0.005), AP by 1,437 ± 40% (P < 0.001), and OC by 1,962 ± 337% (P < 0.001). In addition, it increased mineralized nodule formation by 81 ± 14% (P < 0.001) in a dose‐ and time‐dependent fashion. In equimolar dosages, the parent compound, MEPE, had the full activity of the AC‐100 fragment. AC‐100 elicited a comparable response to both IGF‐I and BMP‐2 with respect to proliferation and differentiation of hMS cells. Using gene expression microarray analysis, we demonstrated that AC‐100 increased (by ∼3‐fold) the mRNA for cyclooxgenase‐2 (COX‐2), an inducible enzyme required for prostaglandin synthesis. Moreover, NS‐398, a specific inhibitor of COX‐2 action completely blocked AC‐100‐induced increases in proliferation and differentiation. Thus, AC‐100 has potent anabolic activity on osteoblast precursor cells in vitro and these effects require the induction of COX‐2.

GAIP Interacting Protein C-Terminus Regulates Autophagy and Exosome Biogenesis of Pancreatic Cancer through Metabolic Pathways

GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.

Loss of ERE binding activity by estrogen receptor-α alters basal and estrogen-stimulated bone-related gene expression by osteoblastic cells†

Estrogen receptor (ER)‐α can signal either via estrogen response element (ERE)‐mediated pathways or via alternate pathways involving protein–protein or membrane signaling. We previously demonstrated that, as compared to wild type (WT) controls, mice expressing a mutant ER‐α lacking the ability to bind EREs (non‐classical estrogen receptor knock‐in (NERKI)) display significant impairments in the skeletal response to estrogen. To elucidate the mechanism(s) underlying these in vivo deficits, we generated U2OS cells stably expressing either WT ER‐α or the NERKI receptor. Compared to cells transfected with the control vector, stable expression of ER‐α, even in the absence of E2, resulted in an increase in mRNA levels for alkaline phosphatase (AP, by 400%, P < 0.01) and a decrease in mRNA levels for insulin growth factor‐I (IGF‐I) (by 65%, P < 0.001), with no effects on collagen I (col I) or osteocalcin (OCN) mRNA levels. By contrast, stable expression of the NERKI receptor resulted in the suppression of mRNA levels for AP, col I, OCN, and IGF‐I (by 62, 89, 60, and 70%, P < 0.001). While E2 increased mRNA levels of AP, OCN, col I, and IGF‐I in ER‐α cells, E2 effects in the NERKI cells on AP and OCN mRNA levels were attenuated, with a trend for E2 to inhibit col I mRNA levels. In addition, E2 had no effects on IGF‐I mRNA levels in NERKI cells. Collectively, these findings indicate that ERE signaling plays a significant role in mediating effects of estrogen on osteoblastic differentiation markers and on IGF‐I mRNA levels. J. Cell. Biochem. 103: 896–907, 2008.

Bone marrow stromal cells express two distinct splice variants of ER-α that are regulated by estrogen

Estrogen plays a critical role in bone metabolism in both sexes. While the major action of estrogen is to inhibit bone resorption, it is now clear that early osteoblastic (or stromal) cells are a target for estrogen action, mediating the effects of estrogen on bone formation as well as resorption. However, little is known about the expression or regulation of the estrogen receptor (ER)‐α in these cells. The expression of ER‐α is regulated by a complex set of promoters and ER‐α splice variants are present in different tissues. Thus, we sought to define the ER‐α splice variants and their regulation by estrogen in the mouse bone marrow stromal cell line, ST‐2, which can be induced to differentiate into mature osteoblasts. ST‐2 cells expressed the mRNAs and proteins for both the 66 and 46 kDa forms of ER‐α; the latter lacks the AF‐1 domain and can transduce estrogen signaling in some tissues, while serving as a dominant negative receptor in others. Using primers specific for each of the five 5′‐untranslated exons of ER‐α, we found that ST‐2 cells utilized only the promoters upstream of exons F and C (in contrast to most reproductive tissues, which utilize promoters upstream of virtually all the five exons). Moreover, 17β‐estradiol (10−8 M) treatment of ST‐2 cells markedly diminished levels of the 66 kDa as well as the 46 kDa ER‐α proteins, largely through suppression of the transcript arising from the F1 promoter. These data thus indicate that: (1) bone marrow stromal cells express at least two variants of ER‐α and (2) estrogen down regulates the ER‐α mRNA and protein in these cells.

Temporal expression patterns of BMP receptors and collagen II (B) during periosteal chondrogenesis

Articular cartilage has a limited ability to repair itself. Periosteal grafts have chondrogenic potential and are used clinically to repair defects in articular cartilage. An organ culture model system for in vitro rabbit periosteal chondrogenesis has been established to study the molecular events of periosteal chondrogenesis in vitro. In this model, bone morphogenetic protein‐2 (BMP2) mRNA expression was found to be upregulated in the first 12 h. BMPs usually transduce their signals through a receptor complex that includes type II and either type IA or type IB BMP receptors. Receptors IA and IB play distinct roles during limb development. We have examined the temporal expression patterns for the mRNAs of these receptors using our experimental model. The mRNA expression patterns of these three BMP receptors differed from one another in periosteal explants during chondrogenesis. When these explants were cultured under chondrogenic conditions (agarose suspension with TGF‐β1 added to the media for the first 2 days), the expression of BMPRII mRNA and that of BMPRIA mRNA varied only slightly and persisted over a long time. In contrast, the expression of BMPRIB mRNA was upregulated within 12 h, peaked at day 5, and fell to a level that was barely detected beyond day 21. Moreover, the expression of BMPRIB mRNA preceded that of collagen type IIB mRNAs, a marker for matrix‐depositing chondrocytes. These data support a role for coordinate expression of BMP2 and its receptors early during periosteal chondrogenesis.