Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis.

Endometriosis, a steroid hormone-dependent disease characterized by aberrant activation of estrogen receptor signaling and progesterone resistance, remains intractable because of the complexity of the pathways underlying its manifestation. We previously showed that eutopic endometria of women with endometriosis exhibit lower expression of Krüppel-like factor 9 (KLF9), a progesterone receptor coregulator in the uterus, relative to that of women without disease. Here we examined whether loss of endometrial KLF9 expression causes ectopic lesion establishment using syngeneic wild-type (WT) mice as recipients of endometrial fragments from WT and Klf9 null donors. We found significantly higher incidence of ectopic lesions with Klf9 null than WT endometria 8 weeks after tissue injection into the intraperitoneal cavity. The increased incidence of lesion establishment with Klf9 null endometria was associated with a higher expression ratio of estrogen receptor 2 isoform relative to that of estrogen receptor 1 and attenuated progesterone receptor levels in endometriotic stromal cells. PCR array analyses of Notch and Hedgehog signaling components in ectopic lesions demonstrated up-regulated expression of select genes (Jag 2, Shh, Gli1, and Stil 1) in Klf9 null lesions relative to that in WT lesions. Immunohistochemical analyses showed increased levels of Notch intracellular domain and Sonic Hedgehog proteins in Klf9 null lesions relative to that in WT lesions, confirming pathway activation. WT recipients with Klf9 null lesions displayed lower systemic levels of TNFα and IL-6 and higher soluble TNF receptor 1 than corresponding recipients with WT lesions. Our results suggest that endometrial KLF9 deficiency promotes endometriotic lesion establishment by the coincident deregulation of Notch-, Hedgehog-, and steroid receptor-regulated pathways.

Endometriosis is a benign gynecological condition that causes considerable morbidity due to associated infertility, debilitating pelvic pain and inflammatory dysfunctions. Diet is a highly modifiable risk factor for many chronic diseases, but its contribution to endometriosis has not been extensively investigated, due partly to the paradoxical inverse association between obesity and disease incidence. Nevertheless, chronic exposure to dietary high-fat intake has been linked to greater systemic inflammation and oxidative stress, both features of women with endometriosis. Here, we evaluated the effects of a high-fat diet (HFD) (45% fat kcal) on endometriosis progression using an immunocompetent mouse model where ectopic lesion incidence was induced in wild-type recipients by ip administration of endometrial fragments from transcription factor Krüppel-like factor 9-null donor mice. We show that HFD significantly increased ectopic lesion numbers in recipient mice with no significant weight gain and modifications in systemic ovarian steroid hormone and insulin levels, relative to control diet-fed (17% fat kcal) mice. HFD promotion of lesion establishment was associated with reductions in stromal estrogen receptor 1 isoform and progesterone receptor expression, increased F4/80-positive macrophage infiltration, higher stromal but not glandular epithelial proliferation, and enhanced expression of proinflammatory and prooxidative stress pathway genes. Lesion-bearing HFD-fed mice also displayed higher peritoneal fluid TNFα and elevated local and systemic redox status than control diet-fed counterparts. Our results suggest that HFD intake exacerbates endometriosis outcome in the absence of ovarian dysfunction and insulin resistance in mice and warrants further consideration with respect to clinical management of endometriosis progression and recurrence in nonobese patients.

The ovarian hormones estrogen and progesterone promote uterine receptivity and successful pregnancy through their cognate receptors functioning in concert with context-dependent nuclear coregulators. Previously, we showed that the transcription factor Krüppel-like factor (KLF) 9 is a progesterone receptor (PGR) coactivator in the uterus and that mice null for Klf9 exhibit subfertility and reduced progesterone sensitivity. The highly related family member KLF13 displays increased expression in uteri of pregnant and nonpregnant Klf9 null mice and similarly regulates PGR-mediated transactivation in endometrial stromal cells. However, a uterine phenotype with loss of Klf13 has not been reported. In the present study, we demonstrate that Klf13 deficiency in mice did not compromise female fertility and pregnancy outcome. Klf13 null females had litter sizes, numbers of implanting embryos, uterine morphology, and ovarian steroid hormone production comparable to those of wild-type (WT) counterparts. Further, pregnant WT and Klf13 null females at Day Postcoitum (DPC) 3.5 had similar uterine Pgr, estrogen receptor, and Wnt-signaling component transcript levels. Nuclear levels of KLF9 were higher in Klf13 null than in WT uteri at DPC 3.5, albeit whole-tissue KLF9 protein and transcript levels did not differ between genotypes. The lack of a similar induction of nuclear KLF9 levels in uteri of virgin Klf13((-/-)) mice relative to WT uteri was associated with lower stromal PGR expression. In differentiating human endometrial stromal cells, coincident KLF9/KLF13 knockdown by small interfering RNA targeting reduced decidualization-associated PRL expression, whereas KLF9 and KLF13 knockdowns alone reduced transcript levels of WNT4 and BMP2, respectively. Results suggest that KLF9 and KLF13 functionally compensate in peri-implantation uterus for pregnancy success.

Female reproductive tract pathologies arise largely from dysregulation of estrogen and progesterone receptor signaling leading to aberrant cell proliferation, survival and differentiation. The signaling pathways orchestrated by these nuclear receptors are complex, require the participation of many nuclear proteins serving as key binding partners or targets and involve a range of paracrine and autocrine regulatory circuits. Members of the Krüppel-like family of transcription factors are ubiquitously expressed in reproductive tissues and have been increasingly implicated as critical co-regulators and integrators of steroid hormone actions. Here we explore the involvement of KLF family members in uterine pathology, describe their currently known molecular mechanisms and discuss their potential as targets for therapeutic intervention.