Adherent epithelial cells require interactions with the extracellular matrix for their survival, though the mechanism is ill-defined. In long term cultures of primary mammary epithelial cells, a laminin-rich basement membrane (BM) but not collagen I suppresses apoptosis, indicating that adhesion survival signals are specific in their response (Pullan et al. 1996. J. Cell Sci. 109:631–642). We now demonstrate that the signal from BM is mediated by integrins and requires both the α6 and β1 subunits. In addition, a hormonal signal from insulin or insulin-like growth factors, but not hydrocortisone or prolactin, is necessary to suppress mammary cell apoptosis, indicating that BM and soluble factors cooperate in survival signaling. Insulin induced autophosphorylation of its receptor whether mammary cells were cultured on collagen I or BM substrata. However, both the tyrosine phosphorylation of insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase were enhanced in cells cultured on BM, as was the phosphorylation of the phosphatidylinositol 3-kinase effector, protein kinase B. These results suggest a novel extracellular matrix–dependent restriction point in insulin signaling in mammary epithelial cells. The proximal signal transduction event of insulin receptor phosphorylation is not dependent on extracellular matrix, but the activation of downstream effectors requires adhesion to BM. Since phosphatidylinositol 3-kinase was required for mammary epithelial cell survival, we propose that a possible mechanism for BM-mediated suppression of apoptosis is through its facilitative effects on insulin signaling.

We have examined the role of integrin-extracellular matrix interactions in the morphogenesis of ductal structures in vivo using the developing mouse mammary gland as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and become mitotically inactive. End buds are surrounded by a basement membrane, which we show contains laminin-1 and collagen IV. To address the role of cell-matrix interactions in gland development, pellets containing function-perturbing anti-beta1 integrin, anti-alpha6 integrin, and anti-laminin antibodies respectively were implanted into mammary glands at puberty. Blocking beta1 integrins dramatically reduced both the number of end buds per gland and the extent of the mammary ductal network, compared with controls. These effects were specific to the end buds since the rest of the gland architecture remained intact. Reduced development was still apparent after 6 days, but end buds subsequently reappeared, indicating that the inhibition of beta1 integrins was reversible. Similar results were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-alpha6 integrin antibody, suggesting that alpha6 is not the important partner for beta1 in this system. The studies with beta1 integrin were confirmed in a culture model of ductal morphogenesis, where we show that hepatocyte growth factor (HGF)-induced tubulogenesis is dependent on functional beta1 integrins. Thus integrins and HGF cooperate to regulate ductal morphogenesis. We propose that both laminin and beta1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for maintaining end bud structure and function in normal pubertal mammary gland development.

BackgroundThe aims of this study were to: determine the prevalence of pectinate ligament dysplasia (PLD) in populations of Basset hounds (BH), Flatcoated retrievers (FCR) and Dandie Dinmont terriers (DDT) resident in the UK; investigate possible associations between the degree of PLD and age, sex and intraocular pressure (IOP) and; investigate possible associations between IOP and age and sex. Gonioscopy was performed in both eyes of 198 BH, 170 FCR and 95 DDT and the percentage of iridocorneal angle affected by PLD was estimated and classified as unaffected (0 %), mildly affected (<20 %), moderately affected (20–90 %) or severely affected (>90 %). Rebound tonometry was performed bilaterally in the majority of enrolled dogs.ResultsSeventy-six of 198 (38.4 %) BH, 36/170 (21.2 %) FCR and 21/95 (22.1 %) DDT were moderately or severely affected by PLD. The prevalence of PLD was significantly higher in BH than both FCR and DDT. In all breeds there was a significant positive correlation between PLD and age. In the BH only there was a significant association between PLD and sex. In the DDT only there was a weak negative correlation between PLD and IOP and a moderately strong negative correlation between IOP and age.ConclusionsPLD is prevalent and significantly associated with age in all three breeds we investigated. The linear relationship between PLD and age can be explained by the progression of PLD over time which would contribute to the high prevalence of PLD despite widespread screening.

ObjectiveBiologic drug therapies represent a huge advance in the treatment of rheumatoid arthritis (RA). However, very good disease control is achieved in only 30% of patients, making identification of biomarkers of response a research priority. We undertook this study to test our hypothesis that differential DNA methylation patterns may provide biomarkers predictive of response to tumor necrosis factor inhibitor (TNFi) therapy in patients with RA.MethodsAn epigenome‐wide association study was performed on pretreatment whole blood DNA from patients with RA. Patients who displayed good response (n = 36) or no response (n = 36) to etanercept therapy at 3 months were selected. Differentially methylated positions were identified using linear regression. Variance of methylation at differentially methylated positions was assessed for correlation with cis‐acting single‐nucleotide polymorphisms (SNPs). A replication experiment for prioritized SNPs was performed in an independent cohort of 1,204 RA patients.ResultsFive positions that were differentially methylated between responder groups were identified, with a false discovery rate of <5%. The top 2 differentially methylated positions mapped to exon 7 of the LRPAP1 gene on chromosome 4 (cg04857395, P = 1.39 × 10−8 and cg26401028, P = 1.69 × 10−8). The A allele of the SNP rs3468 was correlated with higher levels of methylation for both of the top 2 differentially methylated positions (P = 2.63 × 10−7 and P = 1.05 × 10−6, respectively). Furthermore, the A allele of rs3468 was correlated with European League Against Rheumatism nonresponse in the discovery cohort (P = 0.03; n = 56) and in the independent replication cohort (P = 0.003; n = 1,204).ConclusionWe identify DNA methylation as a potential biomarker of response to TNFi therapy, and we report the association between response and the LRPAP1 gene, which encodes a chaperone of low‐density lipoprotein receptor–related protein 1. Additional replication experiments in independent sample collections are now needed.