Kuang-Hsiang Chuang

Monocyte/macrophage androgen receptor suppresses cutaneous wound healing in mice by enhancing local TNF-α expression

Cutaneous wounds heal more slowly in elderly males than in elderly females, suggesting a role for sex hormones in the healing process. Indeed, androgen/androgen receptor (AR) signaling has been shown to inhibit cutaneous wound healing. AR is expressed in several cell types in healing skin, including keratinocytes, dermal fibroblasts, and infiltrating macrophages, but the exact role of androgen/AR signaling in these different cell types remains unclear. To address this question, we generated and studied cutaneous wound healing in cell-specific AR knockout (ARKO) mice. General and myeloid-specific ARKO mice exhibited accelerated wound healing compared with WT mice, whereas keratinocyte- and fibroblast-specific ARKO mice did not. Importantly, the rate of wound healing in the general ARKO mice was dependent on AR and not serum androgen levels. Interestingly, although dispensable for wound closure, keratinocyte AR promoted re-epithelialization, while fibroblast AR suppressed it. Further analysis indicated that AR suppressed wound healing by enhancing the inflammatory response through a localized increase in TNF-alpha expression. Furthermore, AR enhanced local TNF-alpha expression via multiple mechanisms, including increasing the inflammatory monocyte population, enhancing monocyte chemotaxis by upregulating CCR2 expression, and enhancing TNF-alpha expression in macrophages. Finally, targeting AR by topical application of a compound (ASC-J9) that degrades AR protein resulted in accelerated healing, suggesting a potential new therapeutic approach that may lead to better treatment of wound healing.

Targeting the stromal androgen receptor in primary prostate tumors at earlier stages

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates. The differential proliferation rates, yet with similarly expanded intermediate cell populations, indicated that the prostate stromal AR might play a more dominant role than the epithelial AR to promote primary tumor proliferation at an early stage of tumor. Tissue recombination of human prostate stromal cell lines (WPMY1-v or WPMY1-ARsi) with human prostate cancer epithelial cell lines (PC3-v or PC3-AR9) further demonstrated that the AR might function as a suppressor in epithelial cells and a proliferator in stromal cells in the primary prostate tumors. The dual roles of the AR in prostate epithelium and stroma may require us to reevaluate the target and timing of androgen-deprivation therapy for prostate cancer patients and may suggest a need to develop new drugs to selectively target stromal AR in the primary prostate tumors at earlier stages.

Exome sequencing identifies NMNAT1 mutations as a cause of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is an autosomal recessive retinal dystrophy that manifests with genetic heterogeneity. We sequenced the exome of an individual with LCA and identified nonsense (c.507G>A, p.Trp169*) and missense (c.769G>A, p.Glu257Lys) mutations in NMNAT1, which encodes an enzyme in the nicotinamide adenine dinucleotide (NAD) biosynthesis pathway implicated in protection against axonal degeneration. We also found NMNAT1 mutations in ten other individuals with LCA, all of whom carry the p.Glu257Lys variant.

Neutropenia with impaired host defense against microbial infection in mice lacking androgen receptor

Neutrophils, the major phagocytes that form the first line of cell-mediated defense against microbial infection, are produced in the bone marrow and released into the circulation in response to granulocyte-colony stimulating factor (G-CSF). Here, we report that androgen receptor knockout (ARKO) mice are neutropenic and susceptible to acute bacterial infection, whereas castration only results in moderate neutrophil reduction in mice and humans. Androgen supplement can restore neutrophil counts via stabilizing AR in castrated mice, but not in ARKO and testicular feminization mutant (Tfm) mice. Our results show that deletion of the AR gene does not influence myeloid lineage commitment, but significantly reduces the proliferative activity of neutrophil precursors and retards neutrophil maturation. CXCR2-dependent migration is also decreased in ARKO neutrophils as compared with wild-type controls. G-CSF is unable to delay apoptosis in ARKO neutrophils, and ARKO mice show a poor granulopoietic response to exogenous G-CSF injection. In addition, AR can restore G-CSF–dependent granulocytic differentiation upon transduction into ARKO progenitors. We further found that AR augments G-CSF signaling by activating extracellular signal-regulated kinase 1/2 and also by sustaining Stat3 activity via diminishing the inhibitory binding of PIAS3 to Stat3. Collectively, our findings demonstrate an essential role for AR in granulopoiesis and host defense against microbial infection.

Androgen Receptor Influences on Body Defense System via Modulation of Innate and Adaptive Immune Systems: Lessons from Conditional AR Knockout Mice

Upon insult, such as infection or tissue injury, the innate and adaptive immune systems initiate a series of responses to defend the body. Recent studies from immune cell-specific androgen receptor (AR) knockout mice demonstrated that androgen and its receptor (androgen/AR) play significant roles in both immune regulations. In the innate immunity, androgen/AR is required for generation and proper function of neutrophils; androgen/AR also regulates wound healing processes through macrophage recruitment and proinflammatory cytokine production. In adaptive immunity, androgen/AR exerts suppressive effects on development and activation of T and B cells. Removal of such suppression causes thymic enlargement and excessive export of immature B cells. Altogether, androgen/AR plays distinct roles in individual immune cells, and targeting androgen/AR may help in treatment and management of immune-related diseases.

Androgen receptor regulates expression of skeletal muscle-specific proteins and muscle cell types.

C2C12 myoblasts expressing the androgen receptor (AR) were used to analyze the role of androgen-AR signaling pathway in skeletal muscle development. Marked up-regulation of AR expression was observed in differentiated myotubes. A nuclear run-on transcription assay demonstrated that transcription of the AR gene is increased during skeletal muscle cell differentiation. Regulation of skeletal muscle-specific protein expression by the androgen-AR signaling pathway was further analyzed using quadriceps skeletal muscle from wild-type (WT) and AR knock-out (ARKO) male mice. A histological analysis of quadriceps skeletal muscle indicates no morphological differences between ARKO and WT mice. However, the androgen-AR signaling pathway increases expression of slow-twitch-specific skeletal muscle proteins and downregulates fast-twitch-specific skeletal muscle proteins, resulting in an increase of slow-twitch muscle fiber type cells in quadriceps muscle.

Mouse liver dispersion for the diagnosis of early-stage Fatty liver disease: a 70-sample study.

The accumulation of fat droplets within the liver is an important marker of liver disease. This study assesses gradations of steatosis in mouse livers using crawling waves, which are interfering patterns of shear waves introduced into the liver by external sources. The crawling waves are detected by Doppler ultrasound imaging techniques, and these are analyzed to estimate the shear wave speed as a function of frequency between 200 and 360 Hz. In a study of 70 mice with progressive increases in steatosis from 0% to >60%, increases in steatosis are found to increase the dispersion, or frequency dependence, of shear wave speed. This finding confirms an earlier, smaller study and points to the potential of a scoring system for steatosis based on shear wave dispersion.

MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors.

Vascular invasion provides a direct route for tumor metastasis. The degree to which microRNA (miRNA) expression plays a role in tumor vascular invasion is unclear. Here, we report that miR‐494 is up‐regulated in human hepatocellular carcinoma (HCC) tumors with vascular invasion and can promote HCC cell invasiveness by gene inactivation of multiple invasion‐suppressor miRNAs. Our results show that ten eleven translocation (TET) methylcytosine dioxygenase, predominantly TET1 in HCC cells, is a direct target of miR‐494. The reduced 5′‐hydroxymethylcytosine levels observed in the proximal cytosine‐phosphate‐guanine (CpG) regions of multiple invasion‐suppressor miRNA genes are strongly associated with their transcriptional repression upon miR‐494 overexpression, whereas enforced DNA demethylation can abolish the repression. Furthermore, TET1 knockdown shows a similar effect as miR‐494 overexpression. Conversely, miR‐494 inhibition or enforced TET1 expression is able to restore invasion‐suppressor miRNAs and inhibit miR‐494‐mediated HCC cell invasion. Conclusions: miR‐494 can trigger gene silencing of multiple invasion‐suppressor miRNAs by inhibiting genomic DNA demethylation by direct targeting of TET1, thereby leading to tumor vascular invasion. (Hepatology 2015;62:466–480

Targeting Thymic Epithelia AR Enhances T-Cell Reconstitution and Bone Marrow Transplant Grafting Efficacy

Although thymic involution has been linked to the increased testosterone in males after puberty, its detailed mechanism and clinical application related to T-cell reconstitution in bone marrow transplantation (BMT) remain unclear. By performing studies with reciprocal BMT and cell-specific androgen receptor (AR) knockout mice, we found that AR in thymic epithelial cells, but not thymocytes or fibroblasts, played a more critical role to determine thymic cellularity. Further dissecting the mechanism using cell-specific thymic epithelial cell-AR knockout mice bearing T-cell receptor transgene revealed that elevating thymocyte survival was due to the enhancement of positive selection resulting in increased positively selected T-cells in both male and female mice. Targeting AR, instead of androgens, either via genetic knockout of thymic epithelial AR or using an AR-degradation enhancer (ASC-J9®), led to increased BMT grafting efficacy, which may provide a new therapeutic approach to boost T-cell reconstitution in the future.

IL-6 Mediates Macrophage Infiltration after Irradiation via Up-regulation of CCL2/CCL5 in Non-small Cell Lung Cancer

Radiotherapy is effective in reducing primary tumors, however, it may enhance macrophage infiltration to tumor sites, accelerating tumor progression in several ways. We investigated whether radiation can increase macrophage infiltration into non-small cell lung carcinoma (NSCLC) cells. Analysis of in vitro macrophage (differentiated THP-1 cells) migration to either nonirradiated or irradiated tumor cells showed increased migration to the irradiated tumor cells. Because the IL-6 levels in A549 and H157 cells were significantly increased after irradiation, we then investigated whether this increased IL-6 level contributes to radiation-induced macrophage migration. Radiation-induced macrophage infiltration was reduced when IL-6 was knocked down in tumor cells, indicating a positive IL-6 role in this process. To validate this in vitro result, an orthotopic mouse model was developed using a luciferase-tagged H157siIL-6/scramble control (sc) cell set. After tumors developed, the lungs were irradiated, and infiltration of endogenous macrophages and tail-vein injected fluorescent macrophages to tumor sites was investigated. In both groups, increased macrophage infiltration was observed in H157sc cell-derived xenografts compared to H157siIL-6 cell-derived xenografts, confirming the positive IL-6 role in the radiation-induced macrophage infiltration process. In mechanistic dissection studies, radiation-induced up-regulation of CCL2 and CCL5 by IL-6 was detected, and blocking the action of CCL2/CCL5 molecules significantly reduced the number of migrated macrophages to tumor cells after irradiation. These results demonstrate that targeting the IL-6 signaling or CCL2/CCL5 molecules in combination with conventional radiotherapy potentially blocks undesired radiation-induced macrophage infiltration.