Gut Microbiota Regulates the Interaction between Diet and Genetics to Influence Glucose Tolerance.

Background: Metabolic phenotypes are the result of an intricate interplay between multiple factors, including diet, genotype, and the gut microbiome. Per-Arnt-Sim (PAS) kinase is a nutrient-sensing serine/threonine kinase, whose absence (PASK-/-) protects against triglyceride accumulation, insulin resistance, and weight gain on a high-fat diet; conditions that are associated with dysbiosis of the gut microbiome. Methods: Herein, we report the metabolic effects of the interplay of diet (high fat high sugar, HFHS), genotype (PASK-/-), and microbiome (16S sequencing). Results: Microbiome analysis identified a diet-induced, genotype-independent forked shift, with two discrete clusters of HFHS mice having increased beta and decreased alpha diversity. A "lower" cluster contained elevated levels of Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Defferibacteres, and was associated with increased weight gain, glucose intolerance, triglyceride accumulation, and decreased claudin-1 expression. Genotypic effects were observed within the clusters, lower cluster PASK-/- mice displayed increased weight gain and decreased triglyceride accumulation, whereas upper PASK-/- were resistant to decreased claudin-1. Conclusions: These results confirm previous reports that PAS kinase deficiency can protect mice against the deleterious effects of diet, and they suggest that microbiome imbalances can override protection. In addition, these results support a healthy diet for beneficial microbiome maintenance and suggest microbial culprits associated with metabolic disease.

The nuclear variant of bone morphogenetic protein 2 (nBMP2) is expressed in macrophages and alters calcium response.

We previously identified a nuclear variant of bone morphogenetic protein 2 (BMP2), named nBMP2, that is translated from an alternative start codon. Decreased nuclear localization of nBMP2 in the nBmp2NLStm mouse model leads to muscular, neurological, and immune phenotypes-all of which are consistent with aberrant intracellular calcium (Ca2+) response. Ca2+ response in these mice, however, has yet to be measured directly. Because a prior study suggested impairment of macrophage function in nBmp2NLStm mutant mice, bone marrow derived (BMD) macrophages and splenic macrophages were isolated from wild type and nBmp2NLStm mutant mice. Immunocytochemistry revealed that nuclei of both BMD and splenic macrophages from wild type mice contain nBMP2, while the protein is decreased in nuclei of nBmp2NLStm mutant macrophages. Live-cell Ca2+ imaging and engulfment assays revealed that Ca2+ response and phagocytosis in response to bacterial supernatant are similar in BMD macrophages isolated from naïve (uninfected) nBmp2NLStm mutant mice and wild type mice, but are deficient in splenic macrophages isolated from mutant mice after secondary systemic infection with Staphylococcus aureus, suggesting progressive impairment as macrophages respond to infection. This direct evidence of impaired Ca2+ handling in nBMP2 mutant macrophages supports the hypothesis that nBMP2 plays a role in Ca2+ response.

Per-Arnt-Sim Kinase (PASK) Deficiency Increases Cellular Respiration on a Standard Diet and Decreases Liver Triglyceride Accumulation on a Western High-Fat High-Sugar Diet.

Diabetes and the related disease metabolic syndrome are epidemic in the United States, in part due to a shift in diet and decrease in physical exercise. PAS kinase is a sensory protein kinase associated with many of the phenotypes of these diseases, including hepatic triglyceride accumulation and metabolic dysregulation in male mice placed on a high-fat diet. Herein we provide the first characterization of the effects of western diet (high-fat high-sugar, HFHS) on Per-Arnt-Sim kinase mice (PASK-/-) and the first characterization of both male and female PASK-/- mice. Soleus muscle from the PASK-/- male mice displayed a 2-fold higher oxidative phosphorylation capacity than wild type (WT) on the normal chow diet. PASK-/- male mice were also resistant to hepatic triglyceride accumulation on the HFHS diet, displaying a 2.7-fold reduction in hepatic triglycerides compared to WT mice on the HFHS diet. These effects on male hepatic triglyceride were further explored through mass spectrometry-based lipidomics. The absence of PAS kinase was found to affect many of the 44 triglycerides analyzed, preventing hepatic triglyceride accumulation in response to the HFHS diet. In contrast, the female mice showed resistance to hepatic triglyceride accumulation on the HFHS diet regardless of genotype, suggesting the effects of PAS kinase may be masked.

Personal microbiome analysis improves student engagement and interest in Immunology, Molecular Biology, and Genomics undergraduate courses.

A critical area of emphasis for science educators is the identification of effective means of teaching and engaging undergraduate students. Personal microbiome analysis is a means of identifying the microbial communities found on or in our body. We hypothesized the use of personal microbiome analysis in the classroom could improve science education by making courses more applied and engaging for undergraduate students. We determined to test this prediction in three Brigham Young University undergraduate courses: Immunology, Advanced Molecular Biology Laboratory, and Genomics. These three courses have a two-week microbiome unit and students during the 2016 semester students could submit their own personal microbiome kit or use the demo data, whereas during the 2017 semester students were given access to microbiome data from an anonymous individual. The students were surveyed before, during, and after the human microbiome unit to determine whether analyzing their own personal microbiome data, compared to analyzing demo microbiome data, impacted student engagement and interest. We found that personal microbiome analysis significantly enhanced the engagement and interest of students while completing microbiome assignments, the self-reported time students spent researching the microbiome during the two week microbiome unit, and the attitudes of students regarding the course overall. Thus, we found that integrating personal microbiome analysis in the classroom was a powerful means of improving student engagement and interest in undergraduate science courses.

Gender-based differences in host behavior and gut microbiota composition in response to high fat diet and stress in a mouse model.

Obesity is associated with a high prevalence of mood disorders such as anxiety and depression. Both stress and high fat diet can alter the gut microbiota and contribute to obesity. To examine the interrelationships between obesity, stress, gut microbiota and mood disorders, obesity was induced in mice using a high fat diet, and the mice were subsequently stressed using a chronic unpredictable mild stress protocol. During the experiment, the composition of the gut microbiota was analyzed by 16 S rRNA gene high-throughput sequencing, and anxiety-like behaviors were measured. The results revealed distinct gender differences in the impacts of obesity and stress on anxiety-like behaviors, activity levels, and composition of the gut microbiota. Male mice were more vulnerable to the anxiogenic effects of the high fat diet, and obese male mice showed decreased locomotion activity in response to stress whereas obese female mice did not. In females, stress caused the gut microbiota of lean mice to more closely resemble that of obese mice. Taken together, these results suggest the importance of considering gender as a biological variable in studies on the role of gut microbiota in obesity-related mood disorders.

The BMP2 nuclear variant, nBMP2, is expressed in mouse hippocampus and impacts memory.

The novel nuclear protein nBMP2 is synthesized from the BMP2 gene by translational initiation at an alternative start codon. We generated a targeted mutant mouse, nBmp2NLStm, in which the nuclear localization signal (NLS) was inactivated to prevent nuclear translocation of nBMP2 while still allowing the normal synthesis and secretion of the BMP2 growth factor. These mice exhibit abnormal muscle function due to defective Ca2+ transport in skeletal muscle. We hypothesized that neurological function, which also depends on intracellular Ca2+ transport, could be affected by the loss of nBMP2. Age-matched nBmp2NLStm and wild type mice were analyzed by immunohistochemistry, behavioral tests, and electrophysiology to assess nBMP2 expression and neurological function. Immunohistochemical staining of the hippocampus detected nBMP2 in the nuclei of CA1 neurons in wild type but not mutant mice, consistent with nBMP2 playing a role in the hippocampus. Mutant mice showed deficits in the novel object recognition task, suggesting hippocampal dysfunction. Electrophysiology experiments showed that long-term potentiation (LTP) in the hippocampus, which is dependent on intracellular Ca2+ transport and is thought to be the cellular equivalent of learning and memory, was impaired. Together, these results suggest that nBMP2 in the hippocampus impacts memory formation.

Functional Validation of a Complex Loading Whole Spinal Segment Bioreactor Design.

Intervertebral disk (IVD) degeneration is a prevalent health problem that is highly linked to back pain. To understand the disease and tissue response to therapies, ex vivo whole IVD organ culture systems have recently been introduced. The goal of this work was to develop and validate the design of a whole spinal segment culturing system that loads the disk in complex loading similar to the in vivo condition, while preserving the adjacent endplates and vertebral bodies. The complex loading applied to the spinal segment (flexion-extension (FE), bilateral bending, and compression) was achieved with three pneumatic cylinders rigidly attached to a triangular loading platform. A culture container housed the spinal segment and was attached to the loading mechanism, which allowed for loading of the spinal segment. The dynamic bioreactor was able to achieve physiologic loading conditions with 100 N of applied compression and approximately 2-4 N · m of applied torque. The function of the bioreactor was validated through testing of bovine caudal IVDs with intact endplates and vertebral bodies that were isolated within 2 hrs of death and cultured for 14 days. The resulting IVD cell viability following 14 days of loading was much higher than unloaded control IVDs. The loading system accurately mimicked FE, bilateral bending, and compression motions seen during daily activities. The results indicate that this complex dynamic bioreactor may be appropriate for extended preclinical testing of vertebral-mounted spinal devices and therapies.

Dietary Modulation of Gut Microbiota Contributes to Alleviation of Both Genetic and Simple Obesity in Children.

Gut microbiota has been implicated as a pivotal contributing factor in diet-related obesity; however, its role in development of disease phenotypes in human genetic obesity such as Prader-Willi syndrome (PWS) remains elusive. In this hospitalized intervention trial with PWS (n = 17) and simple obesity (n = 21) children, a diet rich in non-digestible carbohydrates induced significant weight loss and concomitant structural changes of the gut microbiota together with reduction of serum antigen load and alleviation of inflammation. Co-abundance network analysis of 161 prevalent bacterial draft genomes assembled directly from metagenomic datasets showed relative increase of functional genome groups for acetate production from carbohydrates fermentation. NMR-based metabolomic profiling of urine showed diet-induced overall changes of host metabotypes and identified significantly reduced trimethylamine N-oxide and indoxyl sulfate, host-bacteria co-metabolites known to induce metabolic deteriorations. Specific bacterial genomes that were correlated with urine levels of these detrimental co-metabolites were found to encode enzyme genes for production of their precursors by fermentation of choline or tryptophan in the gut. When transplanted into germ-free mice, the pre-intervention gut microbiota induced higher inflammation and larger adipocytes compared with the post-intervention microbiota from the same volunteer. Our multi-omics-based systems analysis indicates a significant etiological contribution of dysbiotic gut microbiota to both genetic and simple obesity in children, implicating a potentially effective target for alleviation. RESEARCH IN CONTEXT: Poorly managed diet and genetic mutations are the two primary driving forces behind the devastating epidemic of obesity-related diseases. Lack of understanding of the molecular chain of causation between the driving forces and the disease endpoints retards progress in prevention and treatment of the diseases. We found that children genetically obese with Prader-Willi syndrome shared a similar dysbiosis in their gut microbiota with those having diet-related obesity. A diet rich in non-digestible but fermentable carbohydrates significantly promoted beneficial groups of bacteria and reduced toxin-producers, which contributes to the alleviation of metabolic deteriorations in obesity regardless of the primary driving forces.

Targeted Mutation of Nuclear Bone Morphogenetic Protein 2 Impairs Secondary Immune Response in a Mouse Model.

We recently identified a nuclear variant of the BMP2 growth factor, called nBMP2. In an effort to understand the function of this variant protein, we generated a mouse line in which BMP2 is expressed and functions normally, but nBMP2 is excluded from the nucleus. This novel mutation allows the study of nBMP2 without compromising BMP2 function. To determine whether nBMP2 plays a role in immune function, we performed a series of experiments in which we compared mouse survival, organ weights, immune cells numbers, and bacterial load in wild type and nBmp2NLS(tm) mice following primary and secondary challenges with Staphylococcus aureus. Following primary challenge with S. aureus, wild type and nBmp2NLS(tm) mice showed no differences in survival or bacterial load and generated similar numbers and types of leukocytes, although mutant spleens were smaller than wild type. Secondary bacterial challenge with S. aureus, however, produced differences in survival, with increased mortality seen in nBmp2NLS(tm) mice. This increased mortality corresponded to higher levels of bacteremia in nBmp2NLS(tm) mice and to a reduced enlargement of mutant spleens in response to the secondary infection. Together, these results suggest that the recently described nuclear variant of BMP2 is necessary for efficient secondary immune responses.

Differential responses of gut microbiota to the same prebiotic formula in oligotrophic and eutrophic batch fermentation systems.

The same prebiotics have produced inconsistent effects on microbiota when evaluated in different batch fermentation studies. To understand the reasons behind these discrepancies, we compared impact of one prebiotic formula on the same inoculated fecal microbiota in two frequently used batch systems: phosphate-buffered saline (PBS, oligotrophic) and basal culture medium (BCM, eutrophic). The microbiota was monitored using 454 pyrosequencing. Negative controls (no prebiotic) of both systems showed significant shifts in the microbiota during fermentation, although their pH remained relatively stable, especially in BCM, with increases in Bilophila and Escherichia/Shigella but a decrease in Faecalibacterium. We identified prebiotic responders via redundancy analysis by including both baseline and negative controls. The key positive and negative responders in the two systems were very different, with only 8 consistently modulated OTUs (7 of the 28 positive responders and 1 of the 35 negative responders). Moreover, some OTUs within the same genus responded to the prebiotic in opposite ways. Therefore, to obtain a complete in vitro evaluation of the modulatory effects of a prebiotic on microbiota, it is necessary to use both oligotrophic and eutrophic systems, compare treatment groups with both baseline and negative controls, and analyze the microbiota changes down to the OTU level.

MRI evaluation of spontaneous intervertebral disc degeneration in the alpaca cervical spine.

Animal models have historically provided an appropriate benchmark for understanding human pathology, treatment, and healing, but few animals are known to naturally develop intervertebral disc degeneration. The study of degenerative disc disease and its treatment would greatly benefit from a more comprehensive, and comparable animal model. Alpacas have recently been presented as a potential large animal model of intervertebral disc degeneration due to similarities in spinal posture, disc size, biomechanical flexibility, and natural disc pathology. This research further investigated alpacas by determining the prevalence of intervertebral disc degeneration among an aging alpaca population. Twenty healthy female alpacas comprised two age subgroups (5 young: 2-6 years; and 15 older: 10+ years) and were rated according to the Pfirrmann-grade for degeneration of the cervical intervertebral discs. Incidence rates of degeneration showed strong correlations with age and spinal level: younger alpacas were nearly immune to developing disc degeneration, and in older animals, disc degeneration had an increased incidence rate and severity at lower cervical levels. Advanced disc degeneration was present in at least one of the cervical intervertebral discs of 47% of the older alpacas, and it was most common at the two lowest cervical intervertebral discs. The prevalence of intervertebral disc degeneration encourages further investigation and application of the lower cervical spine of alpacas and similar camelids as a large animal model of intervertebral disc degeneration.

The bamboo-eating giant panda harbors a carnivore-like gut microbiota, with excessive seasonal variations.

UNLABELLED: The giant panda evolved from omnivorous bears. It lives on a bamboo-dominated diet at present, but it still retains a typical carnivorous digestive system and is genetically deficient in cellulose-digesting enzymes. To find out whether this endangered mammalian species, like other herbivores, has successfully developed a gut microbiota adapted to its fiber-rich diet, we conducted a 16S rRNA gene-based large-scale structural profiling of the giant panda fecal microbiota. Forty-five captive individuals were sampled in spring, summer, and late autumn within 1 year. Significant intraindividual variations in the diversity and structure of gut microbiota across seasons were observed in this population, which were even greater than the variations between individuals. Compared with published data sets involving 124 gut microbiota profiles from 54 mammalian species, these giant pandas, together with 9 captive and 7 wild individuals investigated previously, showed extremely low gut microbiota diversity and an overall structure that diverged from those of nonpanda herbivores but converged with those of carnivorous and omnivorous bears. The giant panda did not harbor putative cellulose-degrading phylotypes such as Ruminococcaceae and Bacteroides bacteria that are typically enriched in other herbivores, but instead, its microbiota was dominated by Escherichia/Shigella and Streptococcus bacteria. Members of the class Clostridia were common and abundant in the giant panda gut microbiota, but most of the members present were absent in other herbivores and were not phylogenetically related with known cellulolytic lineages. Therefore, the giant panda appears not to have evolved a gut microbiota compatible with its newly adopted diet, which may adversely influence the coevolutionary fitness of this herbivore. IMPORTANCE: The giant panda, an endangered mammalian species endemic to western China, is well known for its unique bamboo diet. Unlike other herbivores that have successfully evolved anatomically specialized digestive systems to efficiently deconstruct fibrous plant matter, the giant panda still retains a gastrointestinal tract typical of carnivores. We characterized the fecal bacterial communities from a giant panda population to determine whether this animal relies on its symbiotic gut microbiota to cope with the complex carbohydrates that dominate its diet, as is common in other herbivores. We found that the giant panda gut microbiota is low in diversity and highly variable across seasons. It also shows an overall composition typical of bears and entirely differentiated from other herbivores, with low levels of putative cellulose-digesting bacteria. The gut microbiota of this herbivore, therefore, may not have well adapted to its highly fibrous diet, suggesting a potential link with its poor digestive efficiency.

A Filifactor alocis-centered co-occurrence group associates with periodontitis across different oral habitats.

Periodontitis is a highly prevalent polymicrobial disease worldwide, yet the synergistic pattern of the multiple oral pathogens involved is still poorly characterized. Here, saliva, supragingival and subgingival plaque samples from periodontitis patients and periodontally healthy volunteers were collected and profiled with 16S rRNA gene pyrosequencing. Different oral habitats harbored significantly different microbiota, and segregation of microbiota composition between periodontitis and health was observed as well. Two-step redundancy analysis identified twenty-one OTUs, including Porphyromonas gingivalis, Tannerella forsythia and Filifactor alocis, as potential pathogens that were significantly associated with periodontitis and with two periodontitis diagnostic parameters (pocket depth and attachment loss) in both saliva and supragingival plaque habitats. Interestingly, pairwise correlation analysis among the 21 OTUs revealed that Filifactor alocis was positively correlated with seven other putative pathogens (R > 0.6, P < 0.05), forming a co-occurrence group that was remarkably enriched in all three habitats of periodontitis patients. This bacterial cluster showed a higher diagnostic value for periodontitis than did any individual potential pathogens, especially in saliva. Thus, our study identified a potential synergistic ecological pattern involving eight co-infecting pathogens across various oral habitats, providing a new framework for understanding the etiology of periodontitis and developing new diagnoses and therapies.

Biomechanical analysis of the camelid cervical intervertebral disc.

Chronic low back pain (LBP) is a prevalent global problem, which is often correlated with degenerative disc disease. The development and use of good, relevant animal models of the spine may improve treatment options for this condition. While no animal model is capable of reproducing the exact biology, anatomy, and biomechanics of the human spine, the quality of a particular animal model increases with the number of shared characteristics that are relevant to the human condition. The purpose of this study was to investigate the camelid (specifically, alpaca and llama) cervical spine as a model of the human lumbar spine. Cervical spines were obtained from four alpacas and four llamas and individual segments were used for segmental flexibility/biomechanics and/or morphology/anatomy studies. Qualitative and quantitative data were compared for the alpaca and llama cervical spines, and human lumbar specimens in addition to other published large animal data. Results indicate that a camelid cervical intervertebral disc (IVD) closely approximates the human lumbar disc with regard to size, spinal posture, and biomechanical flexibility. Specifically, compared with the human lumbar disc, the alpaca and llama cervical disc size are approximately 62%, 83%, and 75% with regard to area, depth, and width, respectively, and the disc flexibility is approximately 133%, 173%, and 254%, with regard to range of motion (ROM) in axial-rotation, flexion-extension, and lateral-bending, respectively. These results, combined with the clinical report of disc degeneration in the llama lower cervical spine, suggest that the camelid cervical spine is potentially well suited for use as an animal model in biomechanical studies of the human lumbar spine.

A novel bone morphogenetic protein 2 mutant mouse, nBmp2NLS(tm), displays impaired intracellular Ca2+ handling in skeletal muscle.

We recently reported a novel form of BMP2, designated nBMP2, which is translated from an alternative downstream start codon and is localized to the nucleus rather than secreted from the cell. To examine the function of nBMP2 in the nucleus, we engineered a gene-targeted mutant mouse model (nBmp2NLS(tm)) in which nBMP2 cannot be translocated to the nucleus. Immunohistochemistry demonstrated the presence of nBMP2 staining in the myonuclei of wild type but not mutant skeletal muscle. The nBmp2NLS(tm) mouse exhibits altered function of skeletal muscle as demonstrated by a significant increase in the time required for relaxation following a stimulated twitch contraction. Force frequency analysis showed elevated force production in mutant muscles compared to controls from 10 to 60 Hz stimulation frequency, consistent with the mutant muscle's reduced ability to relax between rapidly stimulated contractions. Muscle relaxation after contraction is mediated by the active transport of Ca(2+) from the cytoplasm to the sarcoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA), and enzyme activity assays revealed that SERCA activity in skeletal muscle from nBmp2NLS(tm) mice was reduced to approximately 80% of wild type. These results suggest that nBMP2 plays a role in the establishment or maintenance of intracellular Ca(2+) transport pathways in skeletal muscle.

Publically funded recreation facilities: obesogenic environments for children and families?

Increasing healthy food options in public venues, including recreational facilities, is a health priority. The purpose of this study was to describe the public recreation food environment in British Columbia, Canada using a sequential explanatory mixed methods design. Facility audits assessed policy, programs, vending, concessions, fundraising, staff meetings and events. Focus groups addressed context and issues related to action. Eighty-eighty percent of facilities had no policy governing food sold or provided for children/youth programs. Sixty-eight percent of vending snacks were chocolate bars and chips while 57% of beverages were sugar sweetened. User group fundraisers held at the recreation facilities also sold 'unhealthy' foods. Forty-two percent of recreation facilities reported providing user-pay programs that educated the public about healthy eating. Contracts, economics, lack of resources and knowledge and motivation of staff and patrons were barriers to change. Recreation food environments were obesogenic but stakeholders were interested in change. Technical support, resources and education are needed.

Implementing a whole school physical activity and healthy eating model in rural and remote first nations schools: a process evaluation of action schools! BC.

INTRODUCTION: Aboriginal people who reside in rural and remote areas of Canada often have poorer health than other Canadians. For instance, the prevalence rate of type 2 diabetes is 3 to 5 times higher than for the general population. Chronic disease risk factors such as obesity are also more prevalent. Overweight and obesity have become major health challenges for all Canadian children, but for Aboriginal children, the numbers are 2 to 3 times higher. 'Action Schools! BC' (AS! BC) is a whole-school framework designed as a positive approach to addressing childhood inactivity and unhealthy eating patterns during the school day that was effective for children in a large urban center. The purpose of this study was to explore the feasibility and implementation of AS! BC in 3 remote Aboriginal communities in northern British Columbia. METHODS: The AS! BC model provided tools for schools and teachers to create individualized 'action plans' to increase the opportunities for physical activity (PA) and healthy eating (HE) across 6 'action zones'. These zones included: (1) school environment; (2) scheduled physical education; (3) classroom action; (4) family and community; (5) extra-curricular; and (6) school spirit. Teachers (primarily generalists) were provided with the training and resources necessary to implement their action plan for their class. Schools had three visits from the AS! BC support team. Teachers received specialized training and support, a 'planning guide' and classroom-based resources. Gender- and skill-level-inclusive activities were prioritized. Although the model emphasized choice using a whole-school framework, 'classroom action' was a flagship component. Teachers were asked to provide students with a minimum of 15 additional minutes of PA each school day and at least one HE activity per month in the 'classroom action zone'. Information about implementation was gathered from weekly 'classroom logs' completed by teachers and focus groups with school staff. RESULTS: The logs showed that all 3 schools implemented physical activities (mean = 140 min/week, range = 7-360 min/week) and HE activities (mean = 2.3 times/week, range = 0-10 times/week) but this varied by school and teacher. Adherence to logging was low (34% of eligible weeks). Focus group data showed that the program was well received and that support from the AS! BC master trainer and support team was crucial to delivery of the program. Staff highlighted challenges (eg time, high staff turnover at the schools and lack of financial resources), but felt that with continued support and cultural adaptations they would continue to implement AS! BC in their schools. CONCLUSIONS: The evaluation demonstrated that AS! BC was appropriate and feasible for use in the First Nations schools in these rural and remote communities with some cultural adaptations and ongoing support. Rural and remote locations have very specific challenges that need to be considered in broader dissemination strategies.

Nuclear variants of bone morphogenetic proteins.

BACKGROUND: Bone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. RESULTS: In all three proteins, a bipartite nuclear localization signal (NLS) was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5) containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. CONCLUSIONS: The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

SOX9 mediates the retinoic acid-induced HES-1 gene expression in human breast cancer cells.

We have previously shown that the anti-proliferative effect of retinoic acid in human breast cancer cell line MCF-7 is dependent on HES-1 expression. Here we show that retinoic acid induces HES-1 expression via upregulation of transcription factor SOX9. By expressing a dominant negative form of SOX9, disrupting endogenous SOX9 activity, the retinoic acid-induced HES-1 mRNA expression was inhibited. We found an enhancer regulating HES-1 expression: two SOX9 binding sites upstream of the HES-1 gene that were capable of binding SOX9 in vitro. By performing chromatin immunoprecipitation, we showed that SOX9 binding to the HES-1 enhancer was induced by retinoic acid in vivo. In reporter assays, transfection of a SOX9 expression plasmid increased the activity of the HES-1 enhancer. The enhancer responded to retinoic acid; furthermore, the expression of a dominant negative SOX9 abolished this response. Taken together, we present here a novel transcriptional mechanism in regulating hormone-dependent cancer cell proliferation.

The transcription factor Lc-Maf participates in Col27a1 regulation during chondrocyte maturation.

The transcription factor Lc-Maf, which is a splice variant of c-Maf, is expressed in cartilage undergoing endochondral ossification and participates in the regulation of type II collagen through a cartilage-specific Col2a1 enhancer element. Type XXVII and type XI collagens are also expressed in cartilage during endochondral ossification, and so enhancer/reporter assays were used to determine whether Lc-Maf could regulate cartilage-specific enhancers from the Col27a1 and Col11a2 genes. The Col27a1 enhancer was upregulated over 4-fold by Lc-Maf, while the Col11a2 enhancer was downregulated slightly. To confirm the results of these reporter assays, rat chondrosarcoma (RCS) cells were transiently transfected with an Lc-Maf expression plasmid, and quantitative RT-PCR was performed to measure the expression of endogenous Col27a1 and Col11a2 genes. Endogenous Col27a1 was upregulated 6-fold by Lc-Maf overexpression, while endogenous Col11a2 was unchanged. Finally, in situ hybridization and immunohistochemistry were performed in the radius and ulna of embryonic day 17 mouse forelimbs undergoing endochondral ossification. Results demonstrated that Lc-Maf and Col27a1 mRNAs are coexpressed in proliferating and prehypertrophic regions, as would be predicted if Lc-Maf regulates Col27a1 expression. Type XXVII collagen protein was also most abundant in prehypertrophic and proliferating chondrocytes. Others have shown that mice that are null for Lc-Maf and c-Maf have expanded hypertrophic regions with reduced ossification and delayed vascularization. Separate studies have indicated that Col27a1 may serve as a scaffold for ossification and vascularization. The work presented here suggests that Lc-Maf may affect the process of endochondral ossification by participating in the regulation of Col27a1 expression.