Pten knockout in mouse preosteoblasts leads to changes in bone turnover and strength.

Bone development and remodeling are controlled by the phosphoinositide-3-kinase (Pi3k) signaling pathway. We investigated the effects of downregulation of phosphatase and tensin homolog (Pten), a negative regulator of Pi3k signaling, in a mouse model of Pten deficiency in preosteoblasts. We aimed to identify mechanisms that are involved in the regulation of bone turnover and are linked to bone disorders. Femora, tibiae, and bone marrow stromal cells (BMSCs) isolated from mice with a conditional deletion of Pten (Pten cKO) in Osterix/Sp7-expressing osteoprogenitor cells were compared to Cre-negative controls. Bone phenotyping was performed by µCT measurements, bone histomorphometry, quantification of bone turnover markers CTX and procollagen type 1 N propeptide (P1NP), and three-point bending test. Proliferation of BMSCs was measured by counting nuclei and Ki-67-stained cells. In vitro, osteogenic differentiation capacity was determined by ALP staining, as well as by detecting gene expression of osteogenic markers. BMSCs from Pten cKO mice were functionally different from control BMSCs. Osteogenic markers were increased in BMSCs derived from Pten cKO mice, while Pten protein expression was lower and Akt phosphorylation was increased. We detected a higher trabecular bone volume and an altered cortical bone morphology in Pten cKO bones with a progressive decrease in bone and tissue mineral density. Pten cKO bones displayed fewer osteoclasts and more osteoblasts (P = .00095) per trabecular bone surface and a higher trabecular bone formation rate. Biomechanical analysis revealed a significantly higher bone strength (P = .00012 for males) and elasticity of Pten cKO femora. On the cellular level, both proliferation and osteogenic differentiation capacity of Pten cKO BMSCs were significantly increased compared to controls. Our findings suggest that Pten knockout in osteoprogenitor cells increases bone stability and elasticity by increasing trabecular bone mass and leads to increased proliferation and osteogenic differentiation of BMSCs.

Small integral membrane protein 10 like 1 downregulation enhances differentiation of adipose progenitor cells.

Small integral membrane protein 10 like 1 (SMIM10L1) was identified by RNA sequencing as the most significantly downregulated gene in Phosphatase and Tensin Homologue (PTEN) knockdown adipose progenitor cells (APCs). PTEN is a tumor suppressor that antagonizes the growth promoting Phosphoinositide 3-kinase (PI3K)/AKT/mechanistic Target of Rapamycin (mTOR) cascade. Diseases caused by germline pathogenic variants in PTEN are summarized as PTEN Hamartoma Tumor Syndrome (PHTS). This overgrowth syndrome is associated with lipoma formation, especially in pediatric patients. The mechanisms underlying this adipose tissue dysfunction remain elusive. We observed that SMIM10L1 downregulation in APCs led to an enhanced adipocyte differentiation in two- and three-dimensional cell culture and increased expression of adipogenesis markers. Furthermore, SMIM10L1 knockdown cells showed a decreased expression of PTEN, pointing to a mutual crosstalk between PTEN and SMIM10L1. In line with these observations, SMIM10L1 knockdown cells showed increased activation of PI3K/AKT/mTOR signaling and concomitantly increased expression of the adipogenic transcription factor SREBP1. We computationally predicted an α-helical structure and membrane association of SMIM10L1. These results support a specific role for SMIM10L1 in regulating adipogenesis, potentially by increasing PI3K/AKT/mTOR signaling, which might be conducive to lipoma formation in pediatric patients with PHTS.

PTEN regulates adipose progenitor cell growth, differentiation, and replicative aging.

The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates the insulin signaling pathway. Germline PTEN pathogenic variants cause PTEN hamartoma tumor syndrome (PHTS), associated with lipoma development in children. Adipose progenitor cells (APCs) lose their capacity to differentiate into adipocytes during continuous culture, whereas APCs from lipomas of patients with PHTS retain their adipogenic potential over a prolonged period. It remains unclear which mechanisms trigger this aberrant adipose tissue growth. To investigate the role of PTEN in adipose tissue development, we performed functional assays and RNA-Seq of control and PTEN knockdown APCs. Reduction of PTEN levels using siRNA or CRISPR led to enhanced proliferation and differentiation of APCs. Forkhead box protein O1 (FOXO1) transcriptional activity is known to be regulated by insulin signaling, and FOXO1 was downregulated at the mRNA level while its inactivation through phosphorylation increased. FOXO1 phosphorylation initiates the expression of the lipogenesis-activating transcription factor sterol regulatory element-binding protein 1 (SREBP1). SREBP1 levels were higher after PTEN knockdown and may account for the observed enhanced adipogenesis. To validate this, we overexpressed constitutively active FOXO1 in PTEN CRISPR cells and found reduced adipogenesis, accompanied by SREBP1 downregulation. We observed that PTEN CRISPR cells showed less senescence compared with controls and the senescence marker CDKN1A (p21) was downregulated in PTEN knockdown cells. Cellular senescence was the most significantly enriched pathway found in RNA-Seq of PTEN knockdown versus control cells. These results provide evidence that PTEN is involved in the regulation of APC proliferation, differentiation, and senescence, thereby contributing to aberrant adipose tissue growth in patients with PHTS.

New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction.

In workers of the Western honeybee, Apis mellifera, juvenile hormone (JH) and ecdysteroids regulate many aspects of age polyphenism. Here we investigated whether these derived functions in workers have developed by an uncoupling of endocrine mechanisms in adult queens and workers, or whether parallels can be found between the roles of the two hormones in both castes. We looked at yolk protein metabolism as a process central to the physiology of both queens and workers, and at sperm storage as a feature of the queen alone. Queens of differing fertility status (virgin, virgin but CO2-treated, inseminated, freshly laying and 1-2 years-old) were compared regarding vitellogenin (Vg), JH and ecdysteroid-titers in their hemolymph, as well as ovarian yolk protein and spermathecal gland composition. Our results showed that hormone titres were unrelated to the composition of spermathecal glands. JH-concentrations in the hemolymph were low in the groups of queens characterized by yolk uptake into the ovaries, and high in pre-vitellogenic queens or animals that were forced to interrupt egg-laying by caging. Ecdysteroid-concentrations were higher in untreated virgins than after insemination or during egg-laying. They were not affected by the caging of queens. These patterns of hormone changes were parallel to those known from worker bees. Together, these findings suggest a conserved role for JH as repressor of vitellogenin uptake into tissues, and for ecdysteroids in preparing tissues for this process. An involvement of the two hormones in the regulation of sperm storage seems unlikely. Our results add to the view that JH and ecdysteroids act similarly on the yolk protein metabolism of both castes of A. mellifera. This may imply that it was the biochemical versatility of Vg rather than that of hormonal regulatory circuits that allowed for the functional separation of the two castes.

Human monoclonal IgM antibodies with apoptotic activity isolated from cancer patients.

Monoclonal antibodies are accepted as ideal adjuvant therapeutic reagents for all kinds of diseases. Polyvalent (cross-linking) and low-mutated IgM antibodies (less immunogenic) are believed to be the most effective weapons against cancer. The best sources for these types of antibodies are the cancer patients themselves. Using conventional hybridoma technology, not only are fully human monoclonal IgM antibodies isolated, but also new tumor-related targets can be identified using the same experimental approach. The resulting antibodies can be used directly for therapeutic purposes without further modulation and manipulation. This report describes five newly established human monoclonal IgM antibodies; antibody LM-1 that was isolated from a patient with lung cancer, antibodies PM-1 und PM-2 that were isolated from a patient with pancreatic cancer, and antibodies CM-1 and CM-2 which were isolated from a patient with colon carcinoma. The mainly germ-line encoded antibodies are specific for malignant tissues and show only restricted reactivity with healthy cells. When tested for in vitro functional activity, all five antibodies inhibit tumor cell proliferation of carcinoma cells by inducing apoptosis.

Effects of Pharmacological Agents on Ecdysteroid Synthesis in vitro in Ovaries and Abdominal Integument from Female Adult Crickets, Gryllus bimaculatus de Geer (Ensifera, Gryllidae).

In female adults of Gryllus bimaculatus, ovaries as well as the abdominal integument produce free and conjugated ecdysteroids in vitro (Hoffmann et al., 1992). The aim of the current study was to determine the influence of various potential ecdysteroid biosynthesis effectors (RH 5849, KK 42, diflubenzuron, ketoconazole, azadirachtin, acetylenic and allenic cholesteryl derivatives B1, B6, AL2), and also of the protein synthesis inhibitor cycloheximide, on net release of moulting hormones in vitro by the adult ecdysteroid sources. All the compounds examined can be divided into four groups due to their different effectiveness on both the ecdysiosynthetic tissues. The non-steroidal ecdysteroid agonist RH 5849 (group 1) enhanced ecdysteroid synthesis in ovaries, but inhibited hormone production in the abdominal integument. Treatment in vitro with diflubenzuron (dimilin) and cycloheximide (group 2) strongly reduced ovarian ecdysteroid synthesis whereas they were hardly effective on integumental hormone production. The group 3 compounds (ketoconazole, B1, B6, AL2, azadirachtin A) demonstrated a stronger inhibitory effect on the abdominal integument than on the ovary. The imidazole compound KK 42 (group 4) was a very potent effector of ecdysteroid biosynthesis in both hormone sources.