Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture.

SUMMARY: We isolate and characterize osteoblasts from humans without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders. INTRODUCTION: There is currently no data regarding the expression of specific genes or pathways in human osteoblasts that have not been subjected to extensive in vitro culture. Thus, we developed methods to rapidly isolate progressively enriched osteoblast populations from humans and characterized these cells. METHODS: Needle bone biopsies of the posterior iliac crest were subjected to sequential collagenase digests. The cells from the second digest were stained with an alkaline phosphatase (AP) antibody, and the AP+ cells were isolated using magnetic cell sorting. RESULTS: Relative to AP- cells, the AP+ cells contained virtually all of the mineralizing cells and were enriched for key osteoblast marker genes. The AP+ cells were further purified by depletion of cells expressing CD45, CD34, or CD31 (AP+/CD45/34/31- cells), which represented a highly enriched human osteoblast population devoid of hematopoietic/endothelial cells. These cells expressed osteoblast marker genes but very low to undetectable levels of SOST. We next used high-throughput RNA sequencing to compare the transcriptome of the AP+/CD45/34/31- cells to human fibroblasts and identified genes and pathways expressed only in human osteoblasts in vivo, but not in fibroblasts, including 448 genes unique to human osteoblasts. CONCLUSIONS: We provide a detailed characterization of highly enriched human osteoblast populations without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders.

TIEG-null mice display an osteopenic gender-specific phenotype.

TGFbeta inducible early gene-1 (TIEG) was originally cloned from human osteoblasts (OB) and has been shown to play an important role in TGFbeta/Smad signaling, regulation of gene expression and OB growth and differentiation. To better understand the biological role of TIEG in the skeleton, we have generated congenic TIEG-null (TIEG(-/-)) mice in a pure C57BL/6 background. Through the use of DXA and pQCT analysis, we have demonstrated that the femurs and tibias of two-month-old female TIEG(-/-) mice display significant decreases in total bone mineral content, density, and area relative to wild-type (WT) littermates. However, no differences were observed for any of these bone parameters in male mice. Further characterization of the bone phenotype of female TIEG(-/-) mice involved mechanical 3-point bending tests, micro-CT, and histomorphometric analyses of bone. The 3-point bending tests revealed that the femurs of female TIEG(-/-) mice have reduced strength with increased flexibility compared to WT littermates. Micro-CT analysis of femurs of two-month-old female TIEG(-/-) mice revealed significant decreases in cortical bone parameters compared to WT littermates. Histomorphometric evaluation of the distal femur revealed that female TIEG(-/-) mice also display a 31% decrease in cancellous bone area, which is primarily due to a decrease in trabecular number. At the cellular level, female TIEG(-/-) mice exhibit a 42% reduction in bone formation rate which is almost entirely due to a reduction in double labeled perimeter. Differences in mineral apposition rate were not detected between WT and TIEG(-/-) mice. Taken together, these findings suggest that female TIEG(-/-) mice are osteopenic mainly due to a decrease in the total number of functional/mature OBs.

Phytoestrogen genistein acts as an estrogen agonist on human osteoblastic cells through estrogen receptors alpha and beta.

Genistein, a soybean isoflavone, has estrogen-like activity in mammals, including the prevention of bone loss. However, whether its mechanism of action on bone turnover is distinct from that of estrogen or raloxifene is unknown. Although genistein has been reported to bind both estrogen receptor (ER) isoforms (alpha and beta), little is known concerning differential activation of gene expression via these ER isoforms. To examine this question, comparison of the responses of normal fetal osteoblast (hFOB) cells stably expressing either ERalpha (hFOB/ERalpha9) or ERbeta (hFOB/ERbeta6), to treatment with genistein, 17beta-estradiol (E(2)) or raloxifene were conducted. In hFOB/ERalpha9 cells, both genistein and E(2) increased the endogenous gene expression of the progesterone receptor (PR), the proteoglycan versican, and alkaline phosphatase (AP), but inhibited osteopontin (OP) gene expression and interleukin-6 (IL-6) protein levels. Raloxifene had no effect on these bone markers. Genistein, but not raloxifene, also mimicked E(2) action in the hFOB/ERbeta6 cells increasing PR gene expression and inhibiting IL-6 production. To determine whether the gene regulatory actions of genistein in human osteoblast cells occur at the level of transcription, its action on the transcriptional activity of a PR-A promoter-reporter construct was assessed. Both genistein and E(2) were found to stimulate the PR promoter in the hFOB cell line when transiently co-transfected with either ERalpha or ERbeta. Whereas hFOB cell proliferation was unaffected by E(2), raloxifene or genistein at low concentrations, higher concentrations of genistein, displayed significant inhibition. Together, these findings demonstrate that genistein behaves as a weak E(2) agonist in osteoblasts and can utilize both ERalpha and ERbeta.

Mutual antagonism of estrogen receptors alpha and beta and their preferred interactions with steroid receptor coactivators in human osteoblastic cell lines.

Estrogen is a major sex steroid that affects the growth, maintenance, and homeostasis of the skeleton. Two isoforms of the estrogen receptor (ERalpha and ERbeta) mediate the transcriptional effects of estrogen. Although both isoforms of ER are present and functional in some human osteoblast (OB) cell lines, there is minimal information on the differential regulation of transcription by ERalpha and ERbeta homo- or heterodimers. This report demonstrates that ERalpha and ERbeta coexpression decreases the transcriptional capacity (relative to each ER isoform alone) on an estrogen response element-dependent reporter gene in OBs but not in other non-osteoblastic cell lines. These data suggest that ERalpha and ERbeta coexpression can differentially influence the degree of transcriptional activation in certain cell types. Interestingly, the overexpression of the steroid hormone receptor coactivator-1 (SRC1) resulted in preferential transcriptional enhancement by ERbeta as well as coexpressed ERalpha and ERbeta, whereas SRC2 overexpression appeared to preferentially enhance ERalpha transactivation. SRC3 overexpression failed to enhance estrogen-dependent transcription of any ER combination in OBs. Similar overexpression experiments in COS7 cells exhibited preferential enhancement of ERalpha function with all SRCs, including SRC3. Our data also demonstrated that SRC3 mRNA is reduced in osteoblastic cells, suggesting that SRC3 may have only a minor role in these cells. These data suggest that the transactivation capacity of various ER isoforms is both SRC species and cell type dependent.

The COUP-adjacent repressor (CAR) element participates in the tissue-specific expression of the ovalbumin gene.

The ovalbumin (Ov) gene is an excellent model for the study of tissue-specific gene regulation as it is only active in the estrogen-stimulated oviduct. Previous studies have demonstrated that the negative regulatory element (NRE) in the Ov gene 5'-flanking region is responsible for silencing the gene in oviduct in the absence of steroids. Linker scanning analysis defined an element within the NRE designated the COUP-adjacent repressor (CAR) element as a repressor of Ov gene expression. However, the role of the CAR element in non-oviduct tissues has not been addressed. Using transient transfection analysis of various Ov 5'-flanking region constructs into the estrogen-responsive chicken hepatocyte cell line LMH/2A, we demonstrate that Ov gene expression is not induced by estrogen and that an active repressor element exists in the NRE. Deletion analysis indicates that the region from -134 to -87, which includes the CAR element, mediates this repression. Mutation of the CAR element relieves repression, leading to high levels of gene expression. These data support a model where the inhibition of Ov gene expression in non-oviduct cells is a combination of the lack of essential positive factors and the presence of an active repressor, which binds to the CAR element.

Repression of chick multidrug resistance-associated protein 1 (chMRP1) gene expression by estrogen.

Although a number of genes have been identified whose transcriptional activities are stimulated by estrogen, relatively few have been discovered that are repressed. In an effort to determine whether estrogen can directly repress gene expression, attempts were made to identify genes that are direct targets of the estrogen receptor and whose activities are repressed by it. Because the development and differentiation of the chick oviduct are exquisitely dependent upon estrogen, this seemed an appropriate model system for testing this hypothesis. RNA was isolated from estrogen-treated and estrogen-withdrawn chick oviducts and was subjected to differential display analysis. Surprisingly, one of the products repressed by estrogen encoded the chick homolog of the multidrug resistance-associated protein 1 (MRP1) gene. Further cloning resulted in a chick MRP1 (chMRP1) cDNA clone that is 72% identical with human MRP1. Translation of the chMRP1 sequence indicates a 77% amino acid identity with both the human and mouse MRP1 proteins. Treatment of estrogen-withdrawn chicks with 17beta-estradiol decreased chMRP1 mRNA levels to 50% within 30 min and to 70% by 1h, which is comparable to the level observed with chronic repression by estrogen. ChMRP1 mRNA is present in many other tissues, including the heart, lung, brain, kidney, skeletal muscle, and intestine, but is undetectable in the liver. This study indicates that in estrogen-responsive tissues such as chick oviduct, the regulation of chMRP1 gene expression is controlled by estrogen.

Estrogen opposes the apoptotic effects of bone morphogenetic protein 7 on tissue remodeling.

Interactions between estrogen and growth factor signaling pathways at the level of gene expression play important roles in the function of reproductive tissues. For example, estrogen regulates transforming growth factor beta (TGFbeta) in the uterus during the proliferative phase of the mammalian reproductive cycle. Bone morphogenetic protein 7 (BMP-7), a member of the TGFbeta superfamily, is also involved in the development and function of reproductive tissues. However, relatively few studies have addressed the expression of BMP-7 in reproductive tissues, and the role of BMP-7 remains unclear. As part of an ongoing effort to understand how estrogen represses gene expression and to study its interactions with other signaling pathways, chick BMP-7 (cBMP-7) was cloned. cBMP-7 mRNA levels are repressed threefold within 8 h following estrogen treatment in the chick oviduct, an extremely estrogen-responsive reproductive tissue. This regulation occurs at the transcriptional level. Estrogen has a protective role in many tissues, and withdrawal from estrogen often leads to tissue regression; however, the mechanisms mediating regression of the oviduct remain unknown. Terminal transferase-mediated end-labeling and DNA laddering assays demonstrated that regression of the oviduct during estrogen withdrawal involves apoptosis, which is a novel observation. cBMP-7 mRNA levels during estrogen withdrawal increase concurrently with the apoptotic index of the oviduct. Furthermore, addition of purified BMP-7 induces apoptosis in primary oviduct cells. This report demonstrates that the function of BMP-7 in the oviduct involves the induction of apoptosis and that estrogen plays an important role in opposing this function.

Agouti-related maturation and tissue distribution of alpha-Melanocyte Stimulating Hormone in wild-type (AwJ/AwJ) and mutant (Ay/a,a/a) mice.

Because of ectopic overproduction of agouti protein, yellow alleles (A(y) and A(vy)) of the murine agouti gene may secondarily modulate the synthesis, maturation (i.e., acetylation), and/or tissue deployment of alpha-Melanocyte Stimulating Hormone (MSH). We used HPLC to test the hypothesis that A(y)/a mice exhibit altered concentrations of desacetyl-, monoacetyl-, and diacetyl-alpha-MSH in pituitaries, sera, and telogen hair bulbs when compared to black (a/a) mice. We also used RIA to measure total MSH in those same tissues of A(y)a,a/a, and white-bellied agouti (A(wJ)/A(wJ)) mice (Strain C57BL/6J). We found no evidence that A(y)/a mice possessed an imbalance of des-, mono-, and diacetylated alpha-MSH species. However, radioimmunoassay (RIA) analyses of total MSH suggest that wild-type agouti mice (A(wJ)/A(wJ)) exhibited significantly decreased (P < 0.05) tissue levels of total alpha-MSH in pituitaries, sera, and regenerating hair bulbs when compared to those of mutant A(y)/a and a/a mice.