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.

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. MATERIALS AND METHODS: Fifty-nine men (median age, 69 yr) underwent suppression of sex steroids using a gonadotropin-releasing hormone (GnRH) agonist and aromatase blocker and were replaced with testosterone (T; 5 mg/d) and estradiol (E; 37.5 microg/d). After assessment of bone resorption markers (serum C-terminal telopeptide of type I collagen [CTX] and TRACP5b), they were randomized to sex steroid deficiency (-T, -E), E alone (-T, +E), T alone (+T, -E), or both (+T, +E) and restudied 3 wk later. Bone marrow aspirates were obtained to isolate osteoblastic, T, and monocytic cells using magnetic-activated cell sorting. RESULTS: Serum CTX and TRACP5b increased significantly (by 71% and 15%, p < 0.01 and < 0.001, respectively) in the -T, -E group, and these increases occurred despite a 60% suppression of serum FSH levels (p < 0.001) caused by the GnRH agonist. There were significant E (but not T) effects on preventing increases in serum CTx and TRACP levels. There was a nonsignificant trend (p = 0.122) for E to suppress RANKL mRNA levels in bone marrow osteoblastic cells. Changes in mRNA levels for other cytokines (TNF-alpha, interleukin (IL)-1alpha, IL-1beta, IL-1ra, IFN-gamma) in bone marrow cells were not significant. CONCLUSIONS: E has greater suppressive effects on bone resorption than T, and increased bone resorption after sex steroid deficiency can occur independently of changes in FSH secretion. E effects on bone resorption may be mediated by regulation of RANKL production by osteoblastic cells, although further studies using more highly purified cells may reduce the variability of the mRNA measurements and allow for clearer definition of the mediators of sex steroid action in vivo.

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

Estrogen receptor (ER)-alpha 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-alpha 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-alpha or the NERKI receptor. Compared to cells transfected with the control vector, stable expression of ER-alpha, 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-alpha 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.

Characterization of circulating osteoblast lineage cells in humans.

We recently identified circulating osteoblastic cells using antibodies to osteocalcin (OCN) or alkaline phosphatase (AP). We now provide a more detailed characterization of these cells. Specifically, we demonstrate that 46% of OCN positive (OCN(pos)) cells express AP, and 37% also express the hematopoietic/endothelial marker CD34. Using two different anti-OCN antibodies and forward/side light scatter characteristics by flow cytometry, we find that OCN(pos) cells consist of two distinct populations: one population exhibits low forward/side scatter, consistent with a small cell phenotype with low granularity, and a second population has higher forward/side scatter (larger and more granular cell). The smaller, low granularity population also co-expresses CD34, whereas the larger, more granular cells are CD34 negative. Using samples from 26 male subjects aged 28 to 68 years, we demonstrate that the concentration of circulating OCN(pos) cells increases as a function of age (R=0.59, P=0.002). By contrast, CD34(pos) cells tend to decrease with age (R=-0.31, P=0.18); as a consequence, the ratio of OCN(pos):CD34(pos) cells also increase significantly with age (R=0.54, P=0.022). These findings suggest significant overlap between circulating cells expressing OCN and those expressing the hematopoietic/endothelial marker CD34. Further studies are needed to define the precise role of circulating OCN(pos) cells not only in bone remodeling but rather also potentially in the response to vascular injury.

Serum-free media for periosteal chondrogenesis in vitro.

Organ culture studies involving whole explants of periosteum have been useful for studying chondrogenesis, but to date the standard culture model for these explants has required the addition of fetal bovine serum to the media. Numerous investigators have succeeded in culturing chondrocytes and embryonic cells in serum-free conditions but there have been no studies focused on achieving a defined, serum-free media for culturing periosteal explants. The purpose of the present investigation was to determine if whole periosteal explants can be grown and produce cartilage in serum-free conditions, and to define the minimum media supplements that would be conducive to chondrogenesis. 321 periosteal explants were obtained from the medial proximal tibiae of 31 two month-old NZ white rabbits and cultured using a published agarose suspension organ culture model and DMEM for six weeks. The explants were cultured with and without fetal bovine serum or bovine serum albumin and exposed to transforming growth factor beta alone, a combination of growth factors we call ChondroMix (10 ng/ml transforming growth factor beta, 50 ng/ml basic fibroblast growth factor, and 5 microg/ml growth hormone), and/or ITS+ (2.08 microg/ml each of insulin, transferrin, and selenious acid, plus 1.78 microg/ml linoleic acid and 0.42 mg/ml BSA). Maximal chondrogenic stimulation in this study was observed with the combination of ChondroMix and ITS+. However, the minimal requirement to match or exceed the level of chondrogenic stimulation seen in the standard model (TGF-1 in 10% FBS) was achieved simply by the addition of 2.0 microg/ml insulin in 0.1% BSA-containing medium (p < 0.05). Therefore, based on our results, it would be reasonable to assume that insulin is the component in ITS+ responsible for the observed increase in total cartilage growth. Lower concentrations of insulin were not effective, suggesting that the observed effect of insulin requires activation of the IGF-1 receptor.

Effects of loss of steroid receptor coactivator-1 on the skeletal response to estrogen in mice.

Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen (E) action in many tissues, but its role in mediating E effects on bone is unknown. Thus, we assessed the skeletal response to ovariectomy (ovx) and E replacement in SRC-1 knockout (KO) mice compared with wild-type (WT) littermates. Bone mineral density was measured by dual-energy x-ray absorptiometry and peripheral quantitative computed tomography at baseline and after 2 months of sham surgery, ovx, or ovx plus E replacement. Microcomputed tomography and bone histomorphometry were also performed at the end of the study. Both WT and SRC-1 KO mice lost bone at multiple sites after ovx; however, although an estradiol (E(2)) dose of 10 microg/kg.d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis) in the WT mice, it was entirely ineffective in preventing cancellous bone loss at these sites in the SRC-1 KO mice. This E(2) dose was, however, equally effective on cortical bone in the tibia in the SRC-1 KO and WT mice. Moreover, a 4-fold higher dose of E(2) was able to overcome the deficit in E action in cancellous bone in the SRC-1 KO mice. These findings establish that, in mice, loss of SRC-1 leads to skeletal resistance to E predominantly in cancellous bone.

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 Ab's 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.

Induction of CD-RAP mRNA during periosteal chondrogenesis.

Induction of chondrogenesis and maintenance of the chondrocyte phenotype are critical events for autologous periosteal transplantation, which is a viable approach for cartilage repair. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a recently discovered protein that is mainly produced in cartilage. During development, CD-RAP expression starts at the beginning of chondrogenesis and continues throughout cartilage maturation. In order to investigate the involvement of CD-RAP during periosteal chondrogenesis we have determined the nucleotide sequence of the rabbit CD-RAP mRNA and utilized this information to evaluate the temporal and spatial expression pattern of CD-RAP at the mRNA level during chondrogenesis. When the periosteal explants were cultured under chondrogenic conditions, the expression of CD-RAP was induced, as shown by a 40-fold increase in CD-RAP mRNA between days 7 and 10. The temporal expression pattern of CD-RAP closely mimicked that of collagen type IIB mRNA. Also, the CD-RAP mRNA was localized to the matrix forming chondrocytes in the cambium layer of the periosteum by in situ hybridization as indicated by colocalization with collagen type II mRNA and positive safranin O staining. These data suggest a regulatory role of CD-RAP in periosteal chondrogenesis, which is potentially important for both cartilage repair and fracture healing via callus formation.

The spatiotemporal expression of TGF-beta1 and its receptors during periosteal chondrogenesis in vitro.

Transforming growth factor-beta1 (TGF-beta1) has been shown to stimulate chondrogenesis in periosteal explants cultured in agarose suspension. TGF-betas exert their cellular effects through a heteromeric cell membrane receptor complex consisting of TGF-beta type I and type II receptors. In this study, the spatial and temporal expressions of the type I receptor (TbetaR-I), type II receptor (TbetaR-II) and endogenous TGF-beta1 in periosteal explants cultured in vitro were examined using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The temporal changes in the expression of the TbetaR-I and TbetaR-II mRNAs correlated with that of TGF-beta1. Exogenous administration of TGF-beta1 upregulated the expression of both receptors and of the TGF-beta1 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 of TbetaR-I and TbetaR-II simultaneously among the same cells expressing TGF-beta1. TGF-beta1 treatment increased the expression of TGF-beta1, TbetaR-I and TbetaR-II in mesenchymal cells in the cambium layer at 7 days in culture. Small round chondrocytes showed widely distributed immunoreactivity of TGF-beta1, TbetaR-I and TbetaR-II in the 42-day explants treated with TGF-beta1. These observations support the hypothesis that TGF-beta1 regulates the initiation and formation of cartilage during periosteal chondrogenesis.

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-beta1 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 mRNAwas 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.