CSF1R-dependent macrophages control postnatal somatic growth and organ maturation.

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.

Insulin-like growth factor binding protein related protein 1 knockdown attenuates hepatic fibrosis via the regulation of MMPs/TIMPs in mice.

BACKGROUND: Previous research suggested that insulin-like growth factor binding protein related protein 1 (IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix (ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. METHODS: Hepatic fibrosis was induced by thioacetamide (TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog (Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin (α-SMA), transforming growth factor ß 1 (TGFß1), collagen I, MMPs/TIMPs, Sonic Hedgehog (Shh), and glioblastoma family transcription factors (Gli1) were investigated by immunohistochemical staining and Western blotting analysis. RESULTS: We found that hepatic expression of IGFBPrP1, TGFß1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGFß1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. CONCLUSIONS: Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGFß1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.

IGFBP7 Deletion Promotes Hepatocellular Carcinoma.

Activation of IGF signaling is a major oncogenic event in diverse cancers, including hepatocellular carcinoma (HCC). In this setting, the insulin-like growth factor binding protein IGFBP7 inhibits IGF signaling by binding the IGF1 receptor (IGF1R), functioning as a candidate tumor suppressor. IGFBP7 abrogates tumors by inhibiting angiogenesis and inducing cancer-specific senescence and apoptosis. Here, we report that Igfbp7-deficient mice exhibit constitutively active IGF signaling, presenting with proinflammatory and immunosuppressive microenvironments and spontaneous liver and lung tumors occurring with increased incidence in carcinogen-treated subjects. Igfbp7 deletion increased proliferation and decreased senescence of hepatocytes and mouse embryonic fibroblasts, effects that were blocked by treatment with IGF1 receptor inhibitor. Significant inhibition of genes regulating immune surveillance was observed in Igfbp7-/- murine livers, which was associated with a marked inhibition in antigen cross-presentation by Igfbp7-/- dendritic cells. Conversely, IGFBP7 overexpression inhibited growth of HCC cells in syngeneic immunocompetent mice. Depletion of CD4+ or CD8+ T lymphocytes abolished this growth inhibition, identifying it as an immune-mediated response. Our findings define an immune component of the pleiotropic mechanisms through which IGFBP7 suppresses HCC. Furthermore, they offer a genetically based preclinical proof of concept for IGFBP7 as a therapeutic target for immune management of HCC. Cancer Res; 77(15); 4014-25. ©2017 AACR.

Loss of Igfbp7 causes precocious involution in lactating mouse mammary gland.

BACKGROUND: Insulin like growth factors (IGFs) and their binding proteins (IGFBPs) are secreted peptides that play major roles in regulating the normal development and maturation of mammary gland. While Igfbp7 has been shown to decrease breast tumor growth, its role in regulating the normal mammary gland development has not been studied. To this end, we generated Igfbp7-null mice and examined the development and maturation of mammary glands in the virgin, pregnant and lactating animals. RESULTS: We report here that loss of Igfbp7 significantly retards mammary gland development in the virgin animals. More significantly, the pregnant Igfpb7-null glands contained fewer alveolar structures and that during lactation these glands exhibit the morphological changes that are associated with involution. The transcriptome profile of the Igfbp7-null glands on the lactation day 3 revealed a distinct involution-related gene signature compared to the lactating WT glands. Interestingly, we found that the lactating Igfbp7-null glands exhibit increased expression of Stat3 and enhanced activation of (phosphorylated) Stat3, combined with decreased expression of Stat5 suggesting that the absence of Igfbp7 accelerates the onset of involution. We also found that in absence of Igfpb7, the lactating glands contain increased Igfbp5 protein along with decreased expression of IGF-1 Receptor and Akt activation. Finally, we show that during the normal course of involution, Igfbp7 expression is significantly decreased in the mammary gland. CONCLUSION: Our data suggest that loss of Igfbp7 induces precocious involution possibly through diminished cell survival signals. Our findings identify Igfbp7 as major regulator of involution in the mammary gland.

Insulin-like growth factor binding proteins and mammary gland development.

Mammary gland development is dependent upon insulin-like growth factors (IGFs) as survival factors. The actions of the IGFs are modulated by a family of IGF-binding proteins (IGFBP1-6). Expression of the IGFBPs is both time-dependent and cell-specific during both the developmental phases and the involution of the mammary gland. Although studied extensively in vitro, understanding the roles of IGFBPs in vivo has been difficult, largely due to the fact that IGFBP knock-out mice have no dramatic phenotypes. This review examines the evidence from in vitro studies and the attempts to examine in vivo actions utilising models with IGFBP deficiency or over-expression. In vitro studies demonstrate that IGFBPs can act by inhibition of the survival effects of IGFs, as well as by enhancing the effects of IGFs. Because the IGFBPs are found associated with the extracellular matrix, a role for IGFBPs as a reservoir of IGFs or, alternatively as a potential barrier to IGFs, thereby restricting their entry into particular tissues or cellular compartments was postulated. We also provide evidence with respect to the IGF-independent actions of the IGFBPs which include receptors, nuclear localization, and interaction with the extracellular matrix and cell surface proteins including integrins. We believe that recent findings place some of the IGFBPs in a larger family of extracellular proteins, the secreted cysteine-rich protein (CCN) family, which have similar structural domains (involved in binding to IGFs, extracellular matrix and integrins) and are heavily implicated in tissue re-modeling and morphogenesis.

CCN6 (WISP3) decreases ZEB1-mediated EMT and invasion by attenuation of IGF-1 receptor signaling in breast cancer.

During progression of breast cancer, CCN6 protein exerts tumor inhibitory functions. CCN6 is a secreted protein that modulates the insulin-like growth factor-1 (IGF-1) signaling pathway. Knockdown of CCN6 in benign mammary epithelial cells triggers an epithelial to mesenchymal transition (EMT), with upregulation of the transcription factor ZEB1/δEF1. How CCN6 regulates ZEB1 expression is unknown. We hypothesized that CCN6 might regulate ZEB1, EMT and breast cancer invasion by modulating IGF-1 signaling. Exogenously added human recombinant CCN6 protein was sufficient to downregulate ZEB1 mRNA and protein levels in CCN6-deficient (CCN6 KD) HME cells and MDA-MB-231 breast cancer cells. Recombinant CCN6 protein decreased invasion of CCN6 KD cells compared with controls. We discovered that knockdown of CCN6 induced IGF-1 secretion in HME cells cultivated in serum-free medium to higher concentrations than found in MDA-MB-231 cells. Treatment with recombinant CCN6 protein was sufficient to decrease IGF-1 protein and mRNA to control levels, rescuing the effect of CCN6 knockdown. Specific inhibition of IGF-1 receptors using the pharmacological inhibitor NVP-AE541 or short hairpin shRNAs revealed that ZEB1 upregulation due to knockdown of CCN6 requires activation of IGF-1 receptor signaling. Recombinant CCN6 blunted IGF-1-induced ZEB1 upregulation in MDA-MB-231 cells. Our data define a pathway in which CCN6 attenuates IGF-1 signaling to decrease ZEB1 expression and invasion in breast cancer. These results suggest that CCN6 could be a target to prevent or halt breast cancer invasion.

Knockdown of IGF-binding protein 7 inhibits transformation of the endometrial gland in an in vitro model.

Uterine endometrial glands and their secretory products are critical for the implantation and survival of the peri-implantation embryo, and for the establishment of uterine receptivity. We previously reported that insulin-like growth factor binding protein 7 (IGFBP7) is abundantly expressed in uterine glandular epithelial cells during the secretory phase of the menstrual cycle. In the present study, we used a cultured glandular epithelial cell line of human (EM1) to investigate the significance of IGFBP7 in the function of endometrial glands. EM1 cells formed a mesh-like structure on Matrigel, which was accompanied by elevated levels of intracellular cyclic AMP. However, these morphological changes were blocked by treatment with protein kinase A (PKA) inhibitor (H89). IGFBP7 knockdown using specific short interference RNA (siRNA) inhibited the formation of the mesh-like structure on Matrigel. Cyclic AMP analogs, dibutyryl-cAMP, and N(6)-phenyl-cAMP induced the expression of leukemia inhibitory factor (LIF) which is essential for the onset of implantation. Enhanced LIF expression was suppressed by IGFBP7 siRNA treatment. Western blot analysis revealed that IGFBP7 knockdown results in the aberrant, constitutive expression of the MAPK signaling pathway. These results suggest that IGFBP7 regulates morphological changes of glandular cells by interfering with the normal PKA and MAPK signaling pathways that are associated with the transformation and/or differentiation of endometrial glands.

Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries.

Insulin-like growth factor (IGF)-binding protein (IGFBP) 7 is a secreted protein that regulates cellular proliferation, adhesion, and angiogenesis, and has low affinity for IGF compared with that of IGFBP1-IGFBP6. We sought to determine whether IGFBP7 is present in follicular fluid and to elucidate whether IGFBP7 participates in the steroidogenesis of rat mature follicles. Follicular fluid and granulosa cells (GCs) were collected from immature rats 2 days after their treatment with equine chorionic gonadotropin (eCG). IGFBP7 protein was detected in the follicular fluid and the conditioned medium of cultured ovarian GCs by immunoblot analysis. When subconfluent GCs were cultured and treated with FSH and activin, coincubation with FSH and activin markedly increased GC expression of Cyp19a1 (aromatase) mRNA and 17beta-estradiol (E(2)) secretion. The addition of recombinant murine IGFBP7 to these cultures decreased in the activin-enhanced, FSH-stimulated Cyp19a1 mRNA levels in the cells and suppressed the 17beta-E(2) levels in the culture medium. Treatment of GCs with Igfbp7-specific small interfering RNA (siRNA), which knocked down Igfbp7 expression, increased the FSH-stimulated levels of Cyp19a1 but not Cyp11a1 expression. Basal and FSH-stimulated 17beta-E(2) secretion into the culture medium was also enhanced by Igfbp7 siRNA. These results suggest that IGFBP7 suppresses estrogen production in GCs. These observations support the notion that this protein, which is secreted into the follicular fluid, may serve as an intraovarian factor that negatively regulates GC differentiation.

Clinical, radiographic, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy.

A 14-year-old boy presented with a 10-year history of the "sicca" form of seronegative juvenile idiopathic polyarthritis. Severely limited range of motion, pain, and capsular swelling in both small and large weight-bearing joints left him wheelchair-bound. Erythrocyte sedimentation rate and C-reactive protein were normal. Two-phase bone scan revealed tracer uptake of almost every joint at both early and late time points, indicating pathologic exudation and enhanced bone metabolism consistent with severe arthritis. However, radiographic studies revealed no erosive arthropathy but severe osteopenia, dysplastic bone changes, mega os trigonum, and platyspondylia. A magnetic resonance imaging (MRI) scan of the hips showed no signs of synovitis, pannus, or effusion but cartilage irregularities and subchondral cysts. These findings strongly suggested the diagnosis of progressive pseudorheumatoid dysplasia of childhood, an autosomal-recessive disorder of cartilage homeostasis. The patient carries a novel homozygous two-nucleotide deletion in exon 4 of the WISP3 gene. This genetic disorder is an important differential diagnosis of sicca polyarthritis.